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FIRST MAJESTIC SILVER CORP. (AG: NYSE; FR: TSX) (the "Company" or "First Majestic") is pleased to announce the unaudited interim consolidated financial results of the Company for the first quarter ended March 31, 2017. The full version of the financial statements and the management discussion and analysis can be viewed on the Company's web site at http://www.firstmajestic.com or on SEDAR at http://www.sedar.com and on EDGAR at http://www.sec.gov. All amounts are in U.S. dollars unless stated otherwise.
(compared to Fourth Quarter 2016)
- Silver equivalent production decreased 3% to 4.3 million ounces
- Silver production decreased 4% to 2.7 million ounces
- All-in sustaining costs ("AISC") decreased 5% to $12.21 per payable silver ounce
- Revenues increased 4% to $69.1 million
- Realized average silver price increased 3% to $17.55 per ounce
- Mine operating earnings increased 1% to $10.0 million
- Net earnings increased 50% to $2.7 million (Basic EPS of $0.02)
- Adjusted earnings, excluding non-cash and non-recurring items, totaled $3.7 million (Adjusted EPS of $0.02)
- Operating cash flows before working capital and taxes increased 14% to $26.6 million or $0.16 per share (non-GAAP)
- Cash costs increased 3% to $6.68 per payable silver ounce (net of by-product credits)
- Cash and cash equivalents totaled $127.6 million at the end of the quarter


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Colin thrives on connecting with people and help solve business problems. His methods include:
- empowering employee digital transformation
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Need someone to chat with? Schedule time with Colin, virtually of course.

subset: commoncrawl


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The heritage of the WRC...
This Rally, organised by the Automobile Club de Monaco under the High Patronage of Their Serene Highnesses the Prince and Princess of Monaco, is part of the FIA calendar. It is raced over a total length of 2,152
km to 3,572 km, divided into six overall itineraries featuring a total of 17 regularity stages.
Only cars similar to those that competed in Monte-Carlo Rallies until the 51st edition in January 1983 are permitted to enter this competition, ensuring spectators get the chance to see some truly legendary models!
Unlike the WRC Monte-Carlo Rally, the Rallye de Monte-Carlo Historique takes place on open roads and for the duration of the competition, the teams are required to strictly follow the highway code in the countries they pass through.
At the end of the Rally, the penalties accumulated along the route (exceeding the speed limit, failing to follow driving regulations, taking wrong turns, etc.) are converted into points. The final tally is established by adding all those points together, and the team with the lowest number is proclaimed the winner.
On iconic routes…
For the 25th Rallye Historique de Monte-Carlo, the historical vehicles will race along the roads of Europe with the Principality as their ultimate destination.
The drivers and co-pilots will start out from the cities of Oslo, London, Turin, Bad Homburg, and Reims, heading for Monaco.
A great event that will take you back in time to the golden age of motoring.
The itinerary devised for the rally always includes classic stages, most of the generally driven in the past as part of the WRC event.
For the final stage, scheduled for the night of 31 January/1 February, the teams will drive through the famous Col de Turini pass, before returning to Port Hercule in Monaco.
It will be an opportunity to hear the roar of the engines and see the dazzling headlights of these classic vehicles. Nostalgia guaranteed!
To find the event in the Agenda, click HERE.
©Direction de la Communication / ACM Rallye Monte-Carlo Historique


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The time has come when doing an MBA abroad is the best way to boost your professional career. You research the dates to apply to different business schools … Harvard, University of Chicago, Stanford, MIT, Oxford, University of Pennsylvania. They all ask for a competitive score on the GMAT test. You decide to plan your preparation for these exams in advance, so the first thing you want to know is: Where can I take the GMAT exam in Mexico and what do I need to prepare? Next we tell you.
List of Test Centers for the GMAT exam in Mexico
GMAT Mexico City
In Mexico City, there are two places where you can take the GMAT exam: Comprehensive Training Solutions Address: Bosque de Duraznos 65 Despacho 601A, Col. Bosques de las Lomas, 11700
Address: Antonio Dovali Jaime # 70 Torre B, 6th Floor, Alvaro Obregón Delegation, Col. Zedec Santa Fe, 01210
To make sure you get a spot, you will have to book in advance using the official GMAT website. Mexico City is the most popular city to take the GMAT in Mexico, which means that available spots fill up quickly. Book your exam at least two months in advance!
GMAT Monterrey
In Monterrey, you only have one option to take the GMAT exam:
Hidalgo 768 PTE, Colonia Centro, 64000 Monterrey
Reserve your spot.
GMAT Guadalajara
In Guadalajara, there is also only one place to take the test. The address is the following: UNISITE Av. Federalismo Sur # 47, Esquina Pedro Moreno, Colonia Centro, 44100 Guadalajara Reserve your place.
Now the most important question: how do I prepare for the GMAT exam?
We have good and bad news. Let's start with the bad: the GMAT is a VERY difficult test if you are not used to taking standardized tests. It will test your ability to reason under pressure in the area of quantitative reasoning, verbal reasoning and analytical writing. You will be sitting for more than 3 hours with little rest, working at the limit of your ability to reason. The good news is that, as a standardized test, with enough preparation, it is easy to identify and overcome the pitfalls of the test. Exam creators recycle concepts over and over again. In the quantitative section of the GRE, you can expect for example, at least 8 data interpretation problems, 4 geometry, 6 algebra, etc. In Scholastica, We offer GRE and GMAT preparation courses designed by Harvard graduates that prepare you for the most competitive MBAs in the world. Our courses include diagnostic tests, extensive practice material and exercises, and classes taught online in real time. If you are aiming for a competitive graduate degree (top 20), you will need to be in the 90+ percentile. This preparation takes time: you will have to dedicate many hours of study to acquire a good vocabulary in English, to review basic concepts of arithmetic, algebra, geometry and statistics, to develop the ability to read texts under pressure and to identify when an argument is valid or invalid. At Scholastica, we help you prepare to get a competitive score and be admitted to a top MBA.


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"Get your branding right and the customers will come to you," they say. You've probably heard this somewhere before, and it's true. When starting a business, you have to create a brand identity that speaks to your target customers.
An effective brand identity is more than just thinking up a business name, designing a logo, or choosing a color palette. According to Investopedia, brand identity is different from a brand image. It's "how a business presents itself to, and wants to be perceived by, its consumers."
A strong brand identity will set your business apart from your competitors. It will allow you to stand out from the noise, attracting your target market and even audiences outside it.
In this infographic, you'll learn about the key components of branding, the principles of brand identity, and the elements you need to create one. Read on to learn more about creating a strong and effective brand identity.


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We have an optimized web accessible version of this site available. Click here to view.
Looking for a pet-friendly apartment community in Parker, CO? Look no further than Park Sixty Four! As a pet-friendly community, we proudly allow cats and dogs here at Park Sixty Four. Now your pet can relax by your side and enjoy the comforts of your apartment home.
Park Sixty Four proudly partners with PetScreening, a third-party pet application service, to help encourage a pet-responsible environment for all residents and pets. This service makes it easy for pet owners to share their animal records with our team as well as pet groomers, doggy daycares, dog walkers, pet sitters, veterinarians, pet-friendly hotels, and more. All applicants must create a PetScreening account to document animal ownership status. Please call the leasing office for more details.
Pets Welcome Upon Approval.Breed restrictions apply.Limit of 2 pets per home.No weight limit.Pet deposit is $300 for 1 pet, $600 for 2 pets.Non-refundable pet fee is $300 for 1 pet, $600 for 2 pets.Monthly pet rent of $35 will be charged per pet.
Domestic animals, indoor cats only, no exotic animals.Breed restrictions apply; no aggressive breeds. Current vet records, DNA information and pet photo required prior to move-in. Park Sixty Four proudly participates in the PooPrints Pet Waste DNA Program! Ask our leasing agents for full program details today. Pets must be registered with the leasing office and with the PooPrints Program prior to move-in.Please call the leasing office for more details.
If entering multiple e-mail addresses, please insert a comma between them.
Thank you for sharing our community website!
If you are searching for superior apartment home living in Parker, Colorado, you have found it at Park Sixty Four. We are located just a short drive from Interstate 25, enabling seamless travels across the state. Fine dining, superb shopping, and exciting entertainment are also only minutes away from your front door. Our address is the place you want to be.
Residents of Park Sixty Four enjoy spacious one bedroom floor plans, designed perfectly. Our homes are equipped with stellar features such as large walk-in closets, stylish Edison bulb light fixtures, industrial-style built-in shelving, electronic key fob front door access, quartz countertops and a washer and dryer in home. Embrace your Parker surroundings on your patio or balcony. We are a proud pet-friendly community, so don't forget to bring home your pets as well.
From the moment you arrive, you will feel like you have come home. Our professional management team will be delighted to ensure your comfortability. Make life fun again at Park Sixty Four. Schedule a tour today and discover why our community is the best-kept secret in all of Parker, Colorado.


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Avene Hydrance UV Light Hydrating Emulsion SPF30 40ml
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text:
Items 97-116 of 116
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A quality ratchet and full set of sockets is the foundation of any tool box. Eastwood offers tool truck quality hand tools at a fraction of the price tool trucks charge. All Eastwood hand-tools features a lifetime warranty.
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Please enjoy some of the new books and the services the Library is offering this month.
Want to see what books the Library has or order books from another Library in our system? Then check out this how-to guide on creating an account and ordering books using your PSC ID and the SWAN Catalog.
For Women's History month, we recognize the history, activism, and advocacy of American Indian and Indigenous women. Read our blog post, and then check out a book from the Prairie State College Library to learn more!


text:
- 714.521.4811
- firstname.lastname@example.com
|Crow Holdings Industrial
|11260 Cedar Avenue, Bloomington, CA
|Year completed:
|Scope of Services:
|Boundary Survey, Topographic Survey, ALTA Survey, Aerial Survey, Final Parcel Map, Demolition Plan, Onsite Grading Plan, Onsite Storm Drain and Utility Plan, WQMP, SWPPP, Street Improvement Plan, Street Striping Plan, Traffic Control Plan, Construction Staking, Post Construction ALTA Survey
Bloomington Logistics Center is a 35 acre site with 677,000 SF of industrial building and sits just south of Interstate 10 in the Inland Empire. Along with onsite grading and design, Thienes Engineering provided civil engineering and surveying for offsite improvements, including a designated queuing lane off Cedar Avenue with a traffic light at the entrance to the site. This project was completed in 2018.


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San Pedro Plumbing Services
Your Go-To Team for Repair, Maintenance, Installation & More Near Palos Verdes
When you need an experienced plumber for your home or business, call the professionals at Big League Plumbing and Rooter. No matter what issue you are facing, our San Pedro plumbing services are a home run every time.
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text: Wesley understood Christian discipleship more like an apprenticeship to Jesus than a scholarly endeavor where one masters a body of knowledge. So, Wesley developed a process by which Methodists cultivate this kind of discipleship. A process that allowed people who desired this kind of character to be challenged and held accountable for the sake of fleeing the wrath to come. Use this guide with your small group to discuss the class meeting model.


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What Birth of the Nation Has Meant for Mankind
1, 2. (a) Does Jehovah directly answer his own questions at Isaiah 66:9? (b) According to Isaiah 66:10-14, what call does he give?
INSTEAD of answering his own questions recorded at Isaiah 66:9 with a plain No! God moved Isaiah to express this triumphant call:
2 "Rejoice with Jerusalem and be joyful with her, all you lovers of her. Exult greatly with her, all you keeping yourselves in mourning over her; for the reason that you will suck and certainly get satisfaction from the breast of full consolation by her; for the reason that you will sip and experience exquisite delight from the teat of her glory. For this is what Jehovah has said: 'Here I am extending to her peace just like a river and the glory of nations just like a flooding torrent, and you will certainly suck. Upon the flank you will be carried, and upon the knees you will be fondled. Like a man whom his own mother keeps comforting, so I myself shall keep comforting you people; and in the case of Jerusalem you will be comforted. And you will certainly see, and your heart will be bound to exult, and your very bones will sprout just like tender grass. And the hand of Jehovah will certainly be made known to his servants, but he will actually denounce his enemies.'"—Isaiah 66:10-14.
3. Thus the restored Jewish exiles were likened to what, with what enjoyment, and whose hand would they see in this matter?
3 Thus those restored from exile were likened to newborn babes taking nourishment. To the Jewish exiles by the rivers of Babylon the references to sucking the breast and sipping the teat denoted that there would be another Jerusalem built on the site of the destroyed capital city; also that she would mother a population of many citizens or inhabitants and the many other citizens of her realm. (Compare Luke 13:34.) Like mothers then in the Middle East, rebuilt Jerusalem would have her children straddle her left or right thigh, to carry them; and, when seated, she would fondle them lovingly on her lap. Those Jews who loved Jehovah's visible organization back there in the sixth century BCE would rejoice over this, and they would cease from mourning over the fact that for the seventy years of their exile in a pagan land there had been no Jerusalem as the capital city of Jehovah's chosen people. Their joy was like that described in Psalm 126, as in contrast with the sorrow expressed in Psalm 137. Jehovah's servants saw his hand in their deliverance.
4, 5. (a) How did the remnant react to the manifestation of God's "hand" in their behalf, and what did they appreciate more than ever? (b) The time had then arrived for what other action? (c) How does Isaiah describe this?
4 This was no less the case with the members of the spiritual "nation" that was "born" in 1919 CE. After their astonishing liberation from the restrictions imposed during World War I and the threat of their being wiped out, they rejoiced with unbounded joy. Their mourning passed away. More keenly than ever before they came to appreciate that Jehovah is their God and that he has a visible organization, just as he has an invisible organization in the heavens that is like a wife to him. This invisible organization is, for the remnant, a "mother," the "Jerusalem above." (Isaiah 54:1-6; Galatians 4:26) So, coming together again from their disorganized state during World War I, they rejoiced to be nursed, borne along and fondled by this spiritual "mother," the celestial "wife" of God their Father. As "servants" dedicated to him through their older Brother, Jesus Christ, they were comforted to know that the "hand" of Jehovah was operating in their favor. Opposite to this, the time had come for him to "denounce his enemies." This included those who are hostile to the proclaimers of his newborn kingdom, for those who are enemies of his dedicated people are also his own personal enemies. Telling how he will carry out his denunciation, Jehovah goes on to say:
5 "'For here Jehovah himself comes as a very fire, and his chariots are like a storm wind, in order to pay back his anger with sheer rage and his rebuke with flames of fire. For as fire Jehovah himself will for a fact take up the controversy, yes, with his sword, against all flesh; and the slain of Jehovah will certainly become many. Those sanctifying themselves and cleansing themselves for the gardens behind one in the center, eating the flesh of the pig and the loathsome thing, even the jumping rodent, they will all together reach their end,' is the utterance of Jehovah. 'And as regards their works and their thoughts, I am coming in order to collect all the nations and tongues together; and they will have to come and see my glory.'"—Isaiah 66:15-18.
6. So how were the Israelites of Isaiah's day breaking their covenant and befouling themselves?
6 In the days when Isaiah wrote those words, over a century before the destruction of Jerusalem in 607 BCE, the Israelites were grossly violating their national covenant with Jehovah, the law covenant mediated by the prophet Moses. They were sanctifying themselves and cleansing themselves for idolatrous worship in special gardens and were breaking God's laws forbidding them to eat certain things. They defiled themselves and befouled themselves before the God of their covenant and did nothing for his glory.
7. (a) How did Jehovah act in line with his covenant terms to settle the controversy? (b) With whom does God have a like controversy today?
7 So he brought upon those covenant breakers his fiery, stormy judgments, in proper harmony with his covenant terms. He took up the "sword" of execution in settling the controversy that he had with all Jewish flesh. No wonder that merely a few thousand survived the merciless destruction of Jerusalem, only to be dragged off into long exile in pagan idolatrous Babylon. All the surrounding nations and language groups were obliged to witness this manifestation of the glory of Jehovah as the covenant-keeping God who does not lie. All of this solemnly reminds us that the same God has a controversy on a far larger scale with modern-day Christendom, which claims to have replaced ancient Israel as the people of Jehovah God. To his glory he will use his "sword" of vengeance to slay her utterly.
A "Sign" Set Among the Nations
8, 9. (a) To his glory Jehovah has carried out his purpose after having produced what things and after what wartime experience of his people? (b) How did he set out his purpose in Isaiah 66:19, 20?
8 To his international glory this Almighty God has carried out his unalterable purpose as to what would take place after he caused "a land" to be "brought forth with labor pains in one day" and after he caused "a nation" to be "born at one time." As we read his words set out in Isaiah 66:19, 20, we can call to mind what he has gloriously done since the birth of the "nation" made up of the remnant of spiritual Israelites in 1919 after having ceased to be an organized people during World War I, and entertaining little hope or expectation of surviving that world conflict. Here is what he said:
9 "'And I will set among them a sign [ōth, Hebrew; signum, Latin Vulgate], and I will send some of those who are escaped to the nations, to Tarshish, Pul, and Lud, those drawing the bow, Tubal and Javan, the faraway islands, who have not heard a report about me or seen my glory; and they will for certain tell about my glory among the nations. And they will actually bring all your brothers out of all the nations as a gift to Jehovah, on horses and in chariots and in covered wagons and on mules and on swift she-camels, up to my holy mountain, Jerusalem,' Jehovah has said, 'just as when the sons of Israel bring the gift in a clean vessel into the house of Jehovah.'"
10. What has the "sign" that God "set" among the nations proved to be?
10 What has proved to be the "sign" that Jehovah "set" among the nations since World War I? It was a living "sign." Earlier, in Isaiah's prophecy, this mouthpiece of Jehovah said: "Look! I and the children whom Jehovah has given me are as signs and as miracles in Israel from Jehovah of armies, who is residing in Mount Zion." (Isaiah 8:18) In Hebrews 2:11-13 the Christian apostle Paul quotes from those prophetic words and applies them to Jesus Christ and his spirit-begotten disciples, saying: "For both he who is sanctifying and those who are being sanctified all stem from one, and for this cause he is not ashamed to call them 'brothers,' as he says: . . . 'Look! I and the young children, whom Jehovah gave me.'"
11. What have those who make up that "sign" preached worldwide since 1914, and why did they need to be gathered?
11 In agreement with that, the "sign" that Jehovah set up among the nations of earth, for whom "the times of the Gentiles" ran out in 1914, is the spiritual "nation," the members of which have preached worldwide God's kingdom that was born in the heavens at the close of the "appointed times of the nations" in 1914. (Luke 21:24, Authorized Version; New World Translation of the Holy Scriptures; Matthew 24:14) To that end Jehovah gathered together the remnant of spiritual Israelites who had been scattered by the fierce persecution during World War I.
12. (a) Who were to be added to the remnant that had survived World War I? (b) When, especially, did the sending forth of "those who are escaped" begin to take place?
12 Also, there were still others who were to be brought and added to those spiritual Israelites who survived World War I, so as to make up the full membership of the remnant of Christ's spiritual "brothers" at this time. (Matthew 25:40) Notably since the holding of the first general convention of the International Bible Students in Cedar Point, Ohio, USA, in the summer of 1919, Jehovah began to send "some of those who are escaped" of the original remnant "to the nations." More strikingly did he do so at the 1922 convention of the IBSA at the same resort offshore from Sandusky, Ohio, when the president of the Watch Tower Bible and Tract Society climaxed his rousing discourse to the conventioners with the dramatic words: "Advertise, advertise, advertise, the King and his kingdom." Thereafter the congregations of the remnant of spiritual Israelites geared themselves for the proclamation of God's established kingdom, "for a witness to all the nations," by having all members go forth into the field, "from house to house" and publicly. All were ambassadors of the kingdom!—Acts 20:20.
13. In fulfillment of Isaiah's prophecy, who are gathered out of all the nations, and, as it were, by what means faster than by walking?
13 In fulfillment of Isaiah's prophecy, all of this resulted in a grand gathering of the remnant of the spiritual brotherhood, as it were, by all means of conveyance or transportation much faster than by walking. Jehovah had said that he would send "some of those who are escaped" even to far distant lands, and this he did by sending those who had "escaped" alive from the first world war and the famines, pestilences and earthquakes that marked the death-dealing period of 1914-1918 CE, and particularly the persecution that raged against the dedicated Bible students who were cooperating zealously with the Watch Tower Bible and Tract Society worldwide. The persecution in particular caused the spiritual death of some, so that they did not join in the regathering of the surviving remnant after World War I, especially when the call to united action began to be issued through the printed publications of the Society and through its traveling representatives who visited the congregations.
14. About what did the "escaped" ones who were sent forth tell, and even to places in what state of ignorance were they sent?
14 However, the faithful ones did as Jehovah had foretold, "tell about my glory among the nations." (Isaiah 66:19) To all accessible lands and "nations" those obedient "escaped" ones went, even to "faraway islands," telling about the glory of Jehovah's kingdom that had now been established in the heavens in the hands of his reigning King, Jesus Christ. Many in those nations and distant islands had not even heard a report about the true God, Jehovah, and had never discerned that, according to the foretold signs of the times, his glorious kingdom had been born in the heavens.—Revelation 12:1-5, 10.
15. Those spiritual "brothers" who are brought as a "gift to Jehovah" must keep themselves in what spiritual state, and why?
15 All those whom the "escaped" ones brought as their spiritual "brothers" they brought "as a gift to Jehovah," not to some human leader of a religious sect or to some earthly political world power that was vying for popular support. For the "gift" to be acceptable to Jehovah, it had to be clean. That is why the prophecy describes the bringing of the living "gift" as being "just as when the sons of Israel bring the gift in a clean vessel into the house of Jehovah." (Isaiah 66:20) That is why those spiritual "brothers" who make up the "gift" must be no part of this world, yes, they must keep themselves "without spot from the world." (James 1:27) They must keep themselves neutral toward the defiling politics of this unclean system of things. That is why Jehovah said they would be brought "up to my holy mountain, Jerusalem." This does not signify the modern-day Jerusalem, the capital of the political State of Israel. The nation of which it is the capital is 85 percent Jewish and it is a member of the United Nations organization for maintaining world peace and security.
16. Why does Jehovah have nothing in the Middle East today that could be called his "holy mountain"?
16 Modern Jerusalem in the Middle East does not have any "holy mountain" that Jehovah could call his own in a religious sense. Why not? Because what was once his "holy mountain" over there is now crowned by a Muhammadan shrine, the Dome of the Rock. This is for worshipers, not of Jehovah, but of Allah, a deity without a personal name.
17. So what is the "holy mountain, Jerusalem," to which those spiritual "brothers" are brought since the birth of the nation in 1919?
17 All pertinent things being honestly considered, a significant fact becomes evident: In the modern fulfillment of Isaiah 66:20 "my holy mountain" refers to a motherlike organization for spiritual Israelites, namely, "Jerusalem above," spoken of in Galatians 4:26. It is the Zion that has "given birth to her sons," according to Isaiah 66:8. Those "sons" make up the "nation" that was "born at one time," in the postwar year of 1919. In view of that fact, those spiritual "brothers" that were brought by the "escaped" ones since that birth of the "nation" were brought "as a gift to Jehovah" and were put in touch with his royal organization, the "Jerusalem above," this being typified by the rebuilt earthly Jerusalem of old. These being brought forth as her "sons," they were brought into the visible earthly part of Jehovah's universal organization, to be spiritual Jerusalemites.
Restoration of the Pure Worship of Jehovah
18. Since Jehovah promised to take some of the newborn "nation" also "for the priests, for the Levites," what was the most fitting thing for the "nation" to do in their realm of activity?
18 Did that newborn "nation" take up the full worship of the one living and true God, Jehovah, in their postwar realm of spiritual activity? To do so was most appropriate, now that they had been freed from domination by Babylon the Great, the world empire of false religion. And do so they did, in line with the action that Jehovah promised that he would take respecting the newborn "nation," in Isaiah 66:21: "'And from them also I shall take some for the priests, for the Levites,' Jehovah has said." We must remember that, in the case of the Israelites during the seventy years of their exile in ancient Babylon, from 607 to 537 BCE, they were without the active service of the Aaronic high priest and underpriests and of their servants, the Levites, inasmuch as the temple at which they had served was destroyed. With accuracy Jehovah had foretold this, at Hosea 3:4, 5, saying:
19. How had Jehovah foretold the restoration of his worship at Hosea 3:4, 5?
19 "It is because for many days the sons of Israel will dwell . . . without a sacrifice and without a pillar and without an ephod and teraphim. Afterwards the sons of Israel will come back and certainly look for Jehovah their God, and for David their king [the Messiah]; and they will certainly come quivering to Jehovah and to his goodness in the final part of the days."
20. Such action by Jehovah would call for what to be built, and where, and this in behalf of what most vital needs?
20 Thus, besides acting as their Liberator from Babylon, Jehovah would take care of their most vital needs, their spiritual needs. He would reinstate a legitimate high priest along with his underpriests, and these would be assisted by a body of Levites. This would call for the rebuilding of the temple at Jerusalem, at which these could carry on their services so important to the "nation" that would be born "at one time." This would take place in the "land" that would be "brought forth with labor pains in one day," their beloved homeland. Yes, unlikely though this might seem.—Isaiah 66:8.
21. What similar thing took place in a spiritual sense in the case of the newborn "nation" from 1919 onward?
21 In a spiritual sense, the like thing has been true with the remnant of spiritual Israel since Jehovah God liberated them from Babylon the Great (the world empire of false religion) in the postwar year of 1919 CE. He saw to their spiritual needs first. He cleared up their understanding of the office of the Messianic King, Jesus Christ, as High Priest of God. He cleansed them from any religious defilements that still clung to them from association with Babylon the Great and bondage to her. As members of "a chosen race, a royal priesthood, a holy nation, a people for special possession" with regard to the Most High God, they came to appreciate more distinctly than ever before how and why they needed to keep strict neutrality toward the political affairs of this modern world. They saw that the time had come for the one living and true God to 'make a name for himself.' And they awoke to their obligation to exalt God's personal name, Jehovah, above all other names. They discerned their obligation to be witnesses to this God whose name had been overshadowed for centuries, and appropriately adopted the name "Jehovah's witnesses" in the memorable year of 1931.—1 Peter 2:9; Isaiah 43:12; 63:12.
22. Although it involves salvation of creatures, what is the most important issue, the most important teaching of the Bible, and why?
22 Although meriting due consideration in the inspired Bible, the salvation of the human race from sin and death and bondage to Satan the Devil and his organization is not the most important thing, the most important doctrine of the Holy Bible. As the leading article, entitled "Integrity," appearing in the issue of August 15, 1941, of the Watchtower magazine, said: "Jehovah's purpose to have the world ruled by his righteous Government is repeatedly emphasized in the Scriptures. His Government is the Theocracy over which he has made Christ Jesus the King. . . . The primary issue raised by Satan's defiant challenge was and is that of UNIVERSAL DOMINATION." (Paragraphs 1, 19) Hence, the chief purpose of the Most High God is to vindicate this universal sovereignty, which he alone exercises. This he will gloriously do shortly in "the war of the great day of God the Almighty" at the battlefield of Har–Magedon. (Revelation 16:14-16) It is the duty, the privilege, the honor of the "holy nation" of spiritual Israel to maintain its integrity toward Jehovah's universal domination or sovereignty, so proving the Devil a liar.
23. The foiling of what efforts of the enemy nations is in fulfillment of Jehovah's words at Isaiah 66:22?
23 Down to this late date all efforts on the part of the worldly nations of Satan's visible organization to wipe out Jehovah's "nation" that was "born" in 1919 on its newly brought-forth "land" have failed. This is in faithful fulfillment of the assurance given by the Universal Sovereign in these words at Isaiah 66:22: "'For just as the new heavens and the new earth that I am making are standing before me,' is the utterance of Jehovah, 'so the offspring of you people and the name of you people will keep standing.'"
24. (a) The permanence of God's new heavens and new earth is of what assurance to the remnant of spiritual Israel? (b) Of what will the remnant be made a part in due time?
24 Ah, no! Despite all that Satan the Devil and his invisible demons and his visible earthly organization may attempt to do in the near future against them, the remnant of spiritual Israel and its name will not be blotted out. The visible earthly offspring of Satan's organization and their names will not stand much longer. But, in brilliant contrast with that, the offspring of Jehovah's universal organization, the remnant of spiritual Israel, will keep standing in existence; nor will its name be expunged by its enemies and be forgotten. Just as surely as the new heavens and the new earth that the Universal Sovereign Jehovah makes will ever keep standing, so it will be in the case of the remnant of spiritual Israel and its worthy name. This does not mean that the remnant of spiritual Israel will stay on earth forever. No, but in due time they will be taken away and joined to their High Priest, Jesus Christ, in "the new heavens" and will serve as underpriests at the spiritual temple.
25. (a) When did Jehovah start "making" those "new heavens"? (b) Particularly since when have prospective members of the "new earth" made their appearance?
25 Jehovah God began making "the new heavens" at the close of the "appointed times of the nations" in 1914 CE, when he seated his glorified Son Jesus Christ on the heavenly throne, to rule as King of kings and Lord of lords. This is "the Son of David," and, when he was a perfect man on earth, the Universal Sovereign Jehovah made a covenant with him for an everlasting kingdom. (Matthew 21:15) From his celestial throne the King Jesus Christ rules over our terrestrial globe. The making of "the new earth" does not mean or require the destroying of our literal earth. No, for this planet Earth will keep "standing even to time indefinite." (Ecclesiastes 1:4) God's kingdom by Christ will transform it into an everlasting Paradise. Not contradictory to that grand fact, the making of the "new earth" means Jehovah's making of a new human society in full harmony with him and his righteous "new heavens." (2 Peter 3:13) He has already alive on earth millions of prospective members of that "new earth" society. These have made their appearance particularly since the year 1935 CE.
26. (a) How had the coming of this "great crowd" into association with the remnant of spiritual Israelites been referred to in Isaiah 66:12? (b) Where does this "crowd" now serve Jehovah?
26 These have come out of all the nations, races, people and languages and have dedicated themselves to Jehovah God through Christ the High Priest and have symbolized their dedication by getting baptized in water in imitation of their Fine Shepherd, Jesus Christ. (John 10:14, 16) Earlier, in Isaiah 66:12, Jehovah had said that he would extend to the remnant of spiritual Israel, who represent the "Jerusalem above," what he calls "the glory of nations," and this "just like a flooding torrent." Since 1935 this extending of "the glory of nations" to the spiritual remnant has been in progress in the form of the "great crowd" of the "other sheep" of the Fine Shepherd, the reigning King Jesus Christ. (Revelation 7:9-17) Due to their loyalty to Jehovah's Theocratic Government by Christ, the Fine Shepherd has made them "one flock" with the remnant of spiritual Israelites. They are brought to Jehovah's spiritual temple, which is "called even a house of prayer for all the peoples." (Isaiah 56:7; Mark 11:17) At God's spiritual temple for such international prayer the glorious "great crowd" are "rendering him sacred service day and night."—Revelation 7:15.
27, 28. How frequently does the "great crowd" out of "all flesh" meet with the remnant, and what results does their joining in the witness work produce?
27 This marvelous modern development accords with what Jehovah goes on to say, in Isaiah 66:23: "'And it will certainly occur that from new moon to new moon and from sabbath to sabbath all flesh will come in to bow down before me,' Jehovah has said."—Haggai 2:7-9.
28 According to reports, the "great crowd" out of "all flesh" are holding meetings with the remnant of spiritual Israel regularly, "from new moon to new moon [monthly] and from sabbath to sabbath [weekly]," as it were. They are loyally joining with the anointed remnant in the public witnessing and the house-to-house witnessing in order that Jesus' prediction for the "conclusion of the system of things" may be fulfilled: "This good news of the kingdom will be preached in all the inhabited earth for a witness to all the nations." (Matthew 24:3, 14) Those people of "all flesh" who respond to the preaching "come in to bow down" before the Sovereign Jehovah.
29, 30. (a) How does Isaiah 66:24 indicate that those worshipers out of "all flesh" will survive the "great tribulation" with which this system of things ends? (b) Of what will they form the foundation, and, afterward, whom will they lead in the worship of the Maker of things heavenly and earthly?
29 As survivors of the "great tribulation" that lies just ahead for the world, they will witness how Jehovah vindicates his universal sovereignty by destroying his own enemies and their enemies in that worldwide "tribulation." To this effect Isaiah closes his prophecy with the words: "And they [the tribulation survivors] will actually go forth and look upon the carcasses of the men that were transgressing against me; for the very worms upon them will not die and their fire itself will not be extinguished, and they must become something repulsive to all flesh."—Isaiah 66:24.
30 Like the cursed "goats" of Jesus' parable that is fulfilled during this "conclusion of the system of things," those transgressing against the Sovereign Lord Jehovah "will depart into everlasting cutting-off," eternal destruction. (Matthew 24:3; 25:31-46) Then, as the foundation for the "new earth," those sheeplike heirs of the paradise earth will welcome into their midst all those resurrected from the dead to life in the flesh on earth. They will lead all such resurrected flesh in unitedly worshiping the vindicated Maker of the "new heavens" and the "new earth."—Isaiah 65:17; 2 Peter 3:13.
How would you answer the following questions based on Isaiah 66:8-20?
Isa 66 Verse 8: How did the spiritual "nation" of Jehovah come to be "born" into its spiritual "land" in 1919?
Isa 66 Verses 9-14: How did Jerusalem give nourishment and loving care to her "sons"? How does this apply to spiritual sons in our modern day?
Isa 66 Verses 15-18: What practices brought destruction to Jerusalem in 607 BCE, portending what for today?
Isa 66 Verses 19, 20: What "sign" has Jehovah set up "among the nations"? For what purpose?
How would you answer the following questions on Isaiah 66:19-24?
Isa 66 Verse 19: How have "some of those who are escaped" been sent to the "nations"? With what result?
Isa 66 Verse 20: In what way have "the sons of Israel" brought a "gift" to Jehovah's house?
Isa 66 Verse 21: In what way were some taken to serve as "priests"?
Isa 66 Verse 22: Referring to the certainty of "the new heavens and the new earth," what assurance does Jehovah give regarding his restored people?
Isa 66 Verse 23: In what way do we see "all flesh" bowing down before Jehovah "from new moon to new moon and from sabbath to sabbath"?
Isa 66 Verse 24: What will the survivors of the "great tribulation" witness as to the fate of transgressors against Jehovah?
[Picture on page 19]
Like a mother, Jerusalem gave her "sons" loving care
[Picture on page 25]
Having "escaped" from "Babylon the Great," Jehovah's Witnesses go forth with the Kingdom message


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Antalya Land of Legends, known as Disneyland of Turkey, is full of excitement and amazement in every season with its various activities.
Especially if you travel as a family or as a group of friends, this theme park is a perfect place to spend hours in it; since the laughter gets louder when it is shared with the loved ones!
Of course, in the boundaries of The Land of Legends, there will be a lot of laughter, adrenaline, fun and thrill.
Thus, if you want to add some special activities into your Antalya holiday, you can choose to join us to enjoy hilarious hours in The Land of Legends!


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- Home
- Canada Pawn Shops
- Ontario
- Brampton
- Cash Shop
Cash Shop
Cash Shop is a multistore full service pawn shop with 8 stores located in Barrie, Brampton, Mississauga, Toronto. This location can conduct business in English. This pawn store is looking forward to serve you on Sunday as well. Accept Cash. In addition to pawn broking This store also offers payday loans, auto pawn loans, check cashing, money transfer. The particular favorites in This pawn shop are: diamond rings and other jewelry, luxury and high-end watches, TV's, home theatres, iPhones, iPads, Macbooks, car audio, top brand tools like Hilti, Makita, Bosch, work radios, new and vintage game systems, contrlloers and accessories, musical instruments and equipment, stage light, sport stuff, hunting, fishing and camping equipment. The Friendly staff of professionals all eager to assist custmomers on obtaining a loan.
Pawn Value Price Estimator
Use our INSTANT pawn loan estimator to find out how much Cash Shop may pay you for your stuff before even brining it to the store.GET THE PRICE
Pawn Loan Payment Calculator
|Pawn Loan Ammount ($):
|Total Cost ($):
30 Days: 125
60 Days: 130
90 Days: 135
What pawn shop offers:
Payday Loans: Yes
Title Loans: UNKNOWN
Auto Pawn Loans: Yes
High-end Pawn Loans: UNKNOWN
Layaway Program: UNKNOWN
Gemological Laboratory: UNKNOWN
Jewelry Repair: UNKNOWN
Custom Jewelry Design: UNKNOWN
Jewelry Cleaning: UNKNOWN
Watch Repair: UNKNOWN
Watch Battery Replacement: UNKNOWN
Laser Welding: UNKNOWN
Appraising: UNKNOWN
Buy, Sell And Trade: Yes
Custom Car Audio Systems: UNKNOWN
Money Back: UNKNOWN
Trade Ins: UNKNOWN
Insured Protected Loans: UNKNOWN
Private Appointments: UNKNOWN
Negotiable Prices: UNKNOWN
Discounts: UNKNOWN
Warranty: UNKNOWN
Check Cashing: Yes
Money Transfer: Yes
Worst customer service the guy over there from south india is the biggest liar and rude person over phone they say something and when u ask them u should have told on phone he argues and shouts at u and than say I can't tell u my name do whatever u want
Beware when making bitcoins transactions. He did not send the actual currency rate and probably kept the leftover currency in his pocket.
Excellent service. I can definitely advocate that they go out of their way (even on weekends) to ensure a satisfied customer.
Contact Information
|269 Queen St E #5, Brampton, ON L6W 2C2, Canada
|(905) 453-2274
|Open website
Hours of Operation
Monday: 9:45 AM – 8:30 PM
Tuesday: 9:45 AM – 8:30 PM
Wednesday: 9:45 AM – 8:30 PM
Thursday: 9:15 AM – 9:00 PM
Friday: 9:15 AM – 9:00 PM
Saturday: 9:15 AM – 7:00 PM
Sunday: 10:00 AM – 5:00 PM
Best pawn shops in Brampton
Kennedy Cash Shop
(5 stars)
149 Clarence Street, Brampton
Cash M Company
(4.6 stars)
9025 Torbram Road, Brampton
Busters Pawn
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295 Queen Street East Queen Street East, Brampton
Pawn Kings inc
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73 Main St N, Brampton
Green Moolaa Buy Sell Pawn
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4, 263 Queen Street East, Brampton
Cash Shop
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Thamor Money Transfer
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Cash Shop
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269 Queen Street East #5, Brampton
(1.1 stars)
370 Main St. North, Unit 25-B, Brampton
Canada Pawn
(1 stars)
1 Nelson Street West, Brampton


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I hope the guy upstairs doesn't smite me for that title. If I suddenly stop posting here, you'll know what happened. Clearly Tim Tebow has a higher power on his side, because this is getting ridiculous. Another fourth quarter comeback from the
running quarterback. I keep picking against the guy and he keeps ruining my picks. Perhaps I should switch things up, but like I've said for about a month now, teams will figure out how to stop him.
I'm way more upset with the Ravens though. They seem to only get pumped up against playing the Steelers. Since they have already played them twice, maybe I should pick against them for the rest of the season.
Derek's Record Last Week: 11-5
Derek's Overall Record: 100-44
Dylan's Record Last Week: 8-8
Dylan's Overall Record: 39-29
Derek: Because of the Ravens disassociative identity disorder and the injury of Ray Lewis, this is a tough pick for me. The Bengals are no joke and played the Steelers pretty tough last week, but I think John Harbaugh rallies his troops this week and gets a much needed victory before the Harbaugh Bowl.
Winner: Ravens
Dylan: The headline here is that Ray Lewis will be out for the game due to a toe injury. The Ravens are a strong team and even though the Bengals have played pretty well, I think Baltimore will be flying around to make up for the loss of their team leader. The Ravens being at home should help.
Winner: Ravens
Jacksonville Jaguars @ Cleveland Browns
Derek: Blaine Gabbert is probably the worst starting quarterback in the league, and Colt McCoy isn't much better. Both teams have solid defenses, so I'd expect an ugly low-scoring affair. I've seen Frank Gore run on the Browns before, so I'll take Maurice Jones-Drew here.
Winner: Jaguars
Dylan: This game could be the equivalent to taking an Ambien and honestly, either team could come away with this one. However, I'm going to go with Cleveland at home in a close one. Rashean Mathis is out for the season due to a torn ACL, so Colt McCoy could open it up against another weak opponent.
Winner: Browns
Carolina Panthers @ Detroit Lions
Derek: The Lions need a win in the worst way. The Panthers have been awful on defense all year long and I don't have much confidence in them stopping the Lions passing game. This should be a tough game for Cam Newton, who has to go up against a pretty good defense.
Winner: Lions
Dylan: This is my upset pick of the week. I'm basing this off of the fact that Detroit's defensive linemen get back there so quickly, they overpusue and Cam Newton will take advantage. With Detroit at home, this would be a major upset but stranger things have happened. Anyone catch the Thursday night game?
Tampa Bay Buccaneers @ Green Bay Packers
Derek: 10-0
Winner: Packers
Dylan: Aaron Rodgers
Winner: Packers
Buffalo Bills @ Miami Dolphins
Derek: The Bills got leveled by the Cowboys last week, so the Dolphins are a welcome sight for them. The Dolphins have played much better as of late, but I don't see the Bills losing this must win game.
Winner: Bills
Dylan: If the Bills are looking to progress and keep up with New England for the division, they better take down the Dolphins. Ryan Fitzpatrick got his payday, as the Bills organization believe they found their franchise
quarterback. For that to be true, he needs to act like one on a weekly basis.
Winner: Bills
Oakland Raiders @ Minnesota Vikings
Derek: Carson Palmer should only get better as the season improves the more practice time he gets with his new team. The Vikings just lost Antoine Winfield for the season and their pass defense wasn't great before that. Michael Bush may be the best backup running back in the league and the Raiders running game didn't miss a beat last week with him starting.
Winner: Raiders
Dylan: With the Vikings being at home, I think their chances are a little better than the Raiders. If I were a betting man, I would say that Christian Ponder makes less mistakes than Carson Palmer, Adrian Peterson scores a couple touchdowns and Jared Allen harasses the quarterback all day.
Winner: Vikings
Dallas Cowboys @ Washington Redskins
Derek: The Redskins offense and quarterback situation is a mess. Their defense is decent but there is no way they can keep up with the Cowboys.
Winner: Cowboys
Dylan: Who is the quarterback for Washington again?
Winner: Cowboys
Arizona Cardinals @ San Francisco 49ers
Derek: People are only calling this a trap game because this is what you are supposed to call these games. Jim Harbaugh will have the 49ers ready to go and all he has to do is point to the Cardinals-Eagles game as to why the 49ers shouldn't overlook this team.
Winner: 49ers
Dylan: The 49ers are on a mission right now and the Cardinals are the next team unfortunate enough to be in their way. I don't think San Francisco falls victim to complacency; they will not overlook the Cardinals and could potentially pick them apart.
Winner: 49ers
Seattle Seahawks @ St. Louis Rams
Derek: Good news, after going 4-0 last week the NFC West is guaranteed to go 2-2 this week. Unfortunately, that is their only choice barring a tie game or two. Man, wouldn't that be funny? For the sake of the 49ers getting another game up in the NFC West, I'll pick the Rams.
Winner: Rams
Dylan: Seattle has been surprising teams this year with some decent play. However, they are a team that plays to the level of their opponent. That being the case against St. Louis, I think that a weaker Seahawks team shows up and the Rams get the best of them.
Winner: Rams
Tennessee Titans @ Atlanta Falcons
Derek: Mike Smith shouldn't have to make the choice of whether to go for it at his own 30-yard-line in overtime in this game so the Falcons should win here.
Winner: Falcons
Dylan: The Falcons at home are a popular pick. Even with the supposed return of Chris Johnson after last weeks performance and the fact the Julio Jones may miss the game, Atlanta should be the favorite. they have a more put together team and they are still competing for superiority with the division rival Saints.
WInner: Falcons
San Diego Chargers @ Chicago Bears
Derek: The Bears are rolling and the Chargers are in trouble. Poor Takeo Spikes. The guy just cannot catch a break.
Winner: Bears
Dylan: The Bears have been performing well when their offensive line keeps Jay Cutler upright and opens lanes for Matt Forte. Not to mention, Devin Hester has continued to make a strong case for himself as a future Hall of Fame candidate. San Diego is a mess and is notorious for horrendous special teams play, where Chicago has excelled.
Winner: Bears
Philadelphia Eagles @ New York Giants
Derek: Mike Vick may not play with two broken ribs, but the Eagles do have Vince Young. Remember when VInce Young took over the winless Titans a few years ago and won a lot of games? Yeah, that won't happen here.
Winner: Giants
Dylan: The Eagles have been arguably the biggest disappointment of the season. Given the elite roster they tout, their record is poor and their fourth quarter performances have been worse. The Giants showed last week that they are a very good football team, even in the loss to the 49ers. I like the Giants to bounce back at home.
WInner: Giants
Kansas City Chiefs @ New England Patriots
Derek: Time to shave Todd Haley, it's over.
Winner: Patriots
Dylan: No Matt Cassel. No Jamaal Charles. Tom Brady remembers why he missed a season. The division is open for the taking after the Jets colossal meltdown on Thursday Night Football...these are few of many reasons the Patriots will win Monday Night. I wouldn't be surprised to see Ryan Mallett take some snaps in the 4th quarter.
Winner: Patriots


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Increased expression of NGF after spinal cord injury induces sprouting of primary nociceptive axons. Exogenous application of NGF also results in extensive sprouting of these axons and causes chronic pain in uninjured animals. During development, semaphorin3A is thought to act as a repulsive guidance cue for NGF-responsive nociceptive afferents, restricting their projections to the superficial dorsal horn. We investigated the ability of semaphorin3A to selectively reduce NGF-induced sprouting and neuropathic pain in adult rats. The chemorepulsive effect of virus-mediated semaphorin3A expression was shown to counteract the sprouting induced by NGF in a dose-dependent manner, both in vitro and in adult rat spinal cords. Coexpression of semaphorin3A and NGF at moderate to low concentrations within the adult spinal cord reduced sprouting of calcitonin gene-related peptide and substance P-containing axons compared with GFP and NGF coexpression controls. At high expression levels of NGF, there was no difference in sprouting between the semaphorin3A-treated and control groups. The distribution of endogenous primary nociceptive afferents in the spinal cord appeared to be unaffected by semaphorin3A treatment in these experiments. Behavioral assessment shows that semaphorin3A coexpression with NGF led to decreased mechanical allodynia but no significant reductions in thermal hyperalgesia. These findings demonstrate directly that mature sensory afferents maintain their responsiveness to semaphorin3A, suggesting that this molecule might be used therapeutically to control aberrant sensory sprouting involved in pain or autonomic dysfunction.
NGF action on either peripheral or central nerve terminals greatly exacerbates primary sensory nociception. Injury to the spinal cord results in an increased expression of NGF (Nakamura and Bregman, 2001), which acts to increase both neuropeptide expression and sprouting of nociceptive axons (Christensen and Hulsebosch, 1997; Gwak et al., 2003). The relative contribution by each of these potential mechanisms to the type and severity of pain is not known. Several studies demonstrate that administration of antibodies directed against NGF or TrkA-IgG chimera greatly reduce NGF-induced pain responses and also reduce both sprouting and neuropeptide expression (McMahon et al., 1995; Christensen and Hulsebosch, 1997; Gwak et al., 2003). To better understand the components of pain contributed by spouting of nociceptive afferents, we have used semaphorin3A (Sema3A) to control NGF-induced spouting independent of other NGF-mediated events within the noninjured spinal cord.
Previously, we showed that injection of adenovirus encoding NGF (NGF/Ad) into the adult dorsal spinal cord induced robust sprouting of sensory afferents throughout the entire dorsal horn, ventral horn, and the white matter of the lateral funiculus (Romero et al., 2000). Ectopic distribution of these afferents led to hyperalgesia and chronic pain in normal, unlesioned rats. Because during development Sema3A is thought to restrict sensory axon projections (Messersmith et al., 1995), we constructed an adenovirus encoding Sema3A and investigated whether it could function in the adult spinal cord to reduce NGF-induced sprouting and, thus, prevent the aberrant innervation that contributes to abnormal pain.
Semaphorins are a family of secreted and membrane-anchored glycoproteins important in repulsive axon guidance during neuroembryogenesis (Yu and Kolodkin, 1999). Sema3A, one of the best characterized members in this family, is a diffusible molecule that induces growth cone collapse and axon repulsion of several neuronal populations (He and Tessier-Lavigne, 1997; Kolodkin et al., 1997; Takahashi et al., 1999; Tamagnone et al., 1999), through interaction with a neuropilin-1/plexin-A1 receptor complex. One of its roles during development is the establishment of an appropriate pattern of innervation within the dorsal horn of the spinal cord (Luo et al., 1993; Messersmith et al., 1995; Behar et al., 1996; Puschel et al., 1996; Shepherd et al., 1997; Pasterkamp et al., 2000). Sema3A acts to selectively repel axons from a subset of embryonic dorsal root ganglion (DRG) neurons that are of small diameter, NGF responsive, and involved in thermoreception and nociception. Expression of Sema3A in the embryonic ventral horn is thought to restrict the growth of these axons to laminas I and II of the dorsal horn. The first demonstration that adult neurons retain the ability to respond to Sema3A was shown by in vivo expression of Sema3A in adult corneal epithelium, resulting in the repulsion of peripheral trigeminal thermoreceptors and nociceptors from the Sema3A-expressing regions (Tanelian et al., 1997). More recently, small diameter neurons within the adult DRG have been shown to retain their expression of neuropilin-1/plexin A1 receptors (Reza et al., 1999; Gavazzi et al., 2000). Yet, direct demonstration for in vivo responsiveness to Sema3A by adult DRG neurons is absent.
Materials and Methods
Construction of adenoviral vectors. Replication-defective Sema3A-expressing adenovirus (Sema3A/Ad) was constructed as described by He et al. (1998). The coding region of Sema3A with a Myc-epitope tag (a gift from Dr. Corey S. Goodman, University of California at Berkeley, Berkeley, CA) was inserted into the pAdtrack-GFP vector. Recombinant adenovirus was generated using the AdEasy system (He et al., 1998), in which the vector was modified to include a temperature-sensitive mutation (ts125) within the DNA-binding protein of adenovirus (Romero and Smith, 1998). After transfection of 293 cells, virus production was monitored by green fluorescent protein (GFP) expression and plaque formation. Adenovirus was amplified and purified on a cesium–chloride gradient as described (Romero and Smith, 1998). The viral particle to pfu ratio is ∼5:1.
Explant coculture and repulsion assay. An astrocytoma cell line (U373) was transfected/cotransfected with GFP/Ad, NGF/Ad, and Sema3A/Ad for 8 hr at 37°C. The next day, cells were harvested, aggregated, and embedded in collagen gels with embryonic day (E) 10 chicken DRG at a distance of 700–1000 μm, essentially as described by Messersmith et al. (1995). Explants were cultured in DMEM/N2 medium containing 0.5% FBS and 50 ng/ml β-NGF (Invitrogen, Grand Island, NY). Forty-eight hours later, cultures were fixed with 4% paraformaldehyde and stained with crystal violet. Digital images were taken, and the angle between the borders of DRG neurite outgrowth (as diagrammed in Fig. 1C) was measured to quantify the repulsive/attractive influence from the aggregates. Reduction in the degrees of overall neurite growth is reflective of greater chemorepulsion.
Spinal surgery and adenovirus administration. Forty-six adult (250–300 gm) Sprague Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were used in this study. Spinal cord microinjection was performed as described previously (Romero and Smith, 1998; Romero et al., 2000). Briefly, rats were anesthetized and underwent hemilaminectomies at the T13–L1 vertebral segments. As described previously, animals received 100 μg intraperitoneally of a combined solution of rat CD-4 (W3/25) and CD-45 (MRC OX-22) antisera for transient immune suppression. Each animal received eight injections (0.3 μl; 0.5 mm apart and 1.1 mm deep) of individual or combined viral vectors along the L4–L5 dorsal root entry zone. All injections were made using a glass micropippette pulled to an external diameter of 50 μm and beveled so that multiple injections could be made along the entry zone with minimal damage to the cord. The injection depth and location was controlled using a micromanipulator. To remove variation in depth attributable to breathing, the rat was suspended using a spinal harness. A nanoliter injection device (World Precision Instruments, Sarasota, FL) was used to inject a very small, but precise, volume of virus. At each injection site, 0.3 μl of a saline solution containing GFP/Ad (1.25 × 106 pfu/μl), Sema3A/Ad (1.25 × 106 pfu/μl), or a mixture of GFP/Ad and NGF/Ad or Sema3A/Ad and NGF/Ad at ratios of 1:0.04 (1.25 × 106 and 5.0 × 104 pfu/μl), 1:0.1 (1.25 × 106 and 1.25 × 105 pfu/μl), 1:0.2 (1.25 × 106 and 2.5 × 105 pfu/μl), or 1:1 (1.25 × 106 and 1.25 × 106 pfu/μl) was slowly infused (5 nl/sec) into the cord. After each injection, the micropipette was left in place for 1 min before being withdrawn from the spinal cord. Our previous studies demonstrated that this procedure resulted in robust transgene expression distributed consistently throughout the injected area, with little cell death or tissue damage. The adenovirus-mediated transgene expression was primarily localized to astrocytes in the dorsal horn and motor neurons within the ventral horn on the injected side of the spinal cord, peaking between 7 and 14 d after virus administration (Romero and Smith, 1998; Romero et al., 2000). After injections, dorsal musculature was sutured, and the skin incision was closed. All surgery and animal care was done following the Institutional Animal Care and Research Advisory Committee regulations.
Behavioral analysis. Tactile hyperalgesia was evaluated by measuring the nociceptive response to a cutaneous mechanical stimulus, as described by Chaplan et al. (1994). Briefly, rats were placed in a clear plastic chamber on a wire mesh floor. After a habituation period, a series of eight von Frey hairs (0.41, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50, and 15.10 gm; North Coast Medical, Morgan Hill, CA) were applied to the mid-plantar area of the hindpaws following an up-and-down profile (Dixon, 1980; Chaplan et al., 1994). Stimuli for each paw were presented for ∼6–8 sec at intervals of several seconds. The total number of stimuli applied to each paw ranges from five to nine, depending on different response pattern. The 50% withdrawal threshold was calculated to represent the force causing 50% likelihood of withdrawal response. A minimum of four up-and-down trials was performed to identify the 50% withdrawal threshold. Behavioral response to noxious thermal stimuli was measured according to methods described previously (Romero et al., 2000, 2001). Individuals conducting these experiments were always blinded as to the treatment. All animals were tested before adenoviral injection to establish baselines and then 1 week, and 2 weeks after the injection.
Western blot of NGF and Sema3A. In vitro and in vivo expression of NGF and Sema3A was evaluated as described previously (Tanelian et al., 1997; Romero et al., 2000). The in vitro expression of NGF and Sema3A was evaluated 48 hr after virus infection of U373 cells. One milliliter of culture supernatant was precipitated by 0.1 volume of 0.5% sodium deoxycholate and 0.1 volume of TCA. Proteins were pelleted by centrifugation at 14,000 rpm, washed in 80% acetone, dried, and resuspended in 100 μl of Laemmli's buffer. Twenty microliters of each sample were loaded for the Western blot. For in vivo expression of Sema3A/Ad, rats were anesthetized deeply 8–10 d after virus injection. A 6 mm segment of the ipsilateral spinal cord containing the L4- and L5-injected regions was dissected and immediately frozen. The tissue was then homogenized manually with a dounce in 200 μl of 1% SDS in Tris–EDTA buffer with proteinase inhibitors (10 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mm PMSF) and sonicated using a Branson sonifier 450 (VWR Scientific, West Chester, PA). After centrifuging at 14,000 rpm, supernatant was assayed for protein concentration using a BCA kit (Pierce, Rockford, IL), diluted with 3× Laemmli's buffer, and 50 μg of protein was loaded for each sample. After running the sample in either 14% (for NGF) or 10% (for Sema3A) SDS–polyacrylamide gel, proteins were transferred to polyvinylidene difluoride membranes. Membranes were blocked using 5% nonfat dry milk in TBS with 0.05% Tween 20 (TBST). NGF was identified by rabbit anti-NGF antibody (1:500; Accurate, Westbury, NY), and adenovirus-mediated Sema3A expression was detected by mouse anti-Myc 9E10 antibody (1:1000; Developmental Hybrodoma Bank, Iowa City, IA). After a 3 hr incubation in primary antibody, the membranes were washed five times, 10 min in TBST, and incubated in goat anti-rabbit (for NGF) or mouse (for Sema3A) IgG (1:7500; Promega, Madison, WI) conjugated with alkaline phosphatase for 1 hr. Membranes were washed as above and developed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium solutions (Boehringer Mannheim, Indianapolis, IN).
NGF ELISA for spinal cord samples. For animals injected with various concentrations of NGF/Ad, the spinal cords were isolated and frozen as for Western analysis. The tissues were further processed according to the manufacturer's instructions (Promega), with the additional step of pretreating each sample using G-protein agarose to absorb the rodent IgGs. Homogenates were incubated with G-protein agarose (Boehringer Mannheim) overnight at 4°C while shaking. Protein assays were performed, and 100 and 10 μg/ml (100 μl/well) of each sample was used for the assay. After development, 96-well plates were read at 450 nm using a BioTech E12a microplate reader.
B-subunit of cholera toxin tract tracing. Fourteen days after injection of NGF/Ad into the right side of the spinal cord, a solution of B-subunit of cholera toxin (CTB; List Biological Laboratories, Campbell, CA) was injected into the sciatic nerve. This was done bilaterally using a glass micropipette to inject 2 μl of a 1% solution of CTB into each side. Four days after injection, animals were perfused using 4% paraformaldehyde and processed for cryosectioning. Sections were double-labeled for CTB and calcitonin gene-related peptide (CGRP) to examine whether any of the CGRP-positive axons colabeled with CTB. To identify CTB-labeled axons, sections were stained using goat anti-CTB (1:1000; List Biological Laboratories), followed by CGRP staining. Visualization was achieved by tissue incubation in either fluorescent or biotinylated secondary antibodies. Biotin-labeled tissue was further processed using the Vectastain Elite ABC reagents (Vector Laboratories, Burlingame, CA) and developed using two peroxidase substrate kits for brown and blue color (Vector SG). All images were captured using a coolsnap cf. video camera connected to E-800 epifluorescent microscope (Nikon).
Isolectin B4 binding and CGRP immunofluorescence. Cryosections were incubated simultaneously in rabbit anti-CGRP (1:500; Sigma, St. Louis, MO) and biotinylated isolectin B4 (IB4) lectin (1:100; Sigma). Visualization was achieved by incubation in Texas Red-labeled goat anti-rabbit (1:200; Jackson ImmunoResearch, West Grove, PA) and FITC-labeled streptavidin (1:500; Jackson ImmunoResearch). Sections were mounted on slides and coverslipped with 5% propyl-gallate in glycerol for fluorescence microscopy.
Immunocytochemistry and image analysis. All staining and image analyses were done following methods described previously (Romero et al., 2000, 2001). At the end of the study, animals were perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.5. After perfusion, the lumbar spinal cord was removed and postfixed before it was transferred to a 30% sucrose solution for cryoprotection. Thirty-micrometer-thick tissue sections were cut on a cryostat and divided into five alternate sets. The tissue sections were used either immediately for histological analysis or stored in cryoprotectant solution at –20°C until processed. For immunohistochemistry, floating tissue sections were incubated in polyclonal antiserum against rat CGRP (1: 20,000; Sigma) or substance-P (1:10,000; Sigma–RBI, Natick, MA). After visualization using biotinylated secondary antibodies, axonal growth was quantified using MetaMorph Image Analysis software (Universal Imaging, West Chester, PA). Images from two randomly selected regions within the L4 and L5 segments per animal were quantified by first applying a standardized optical density threshold, followed by measuring the area, in square micrometers, occupied by density profiles equal to or greater than the threshold. For each section, the measurements were taken from the dorsal horn lateral to midline and above the plane of the spinal canal.
Statistical analysis. Raw data from DRG growth angle and image analysis were evaluated using Student's t test or one-way ANOVA, followed by a Tukey–Kramer post hoc test to determine significant differences between treatment. Paw withdrawal latencies (PWLs) to thermal stimuli were analyzed using two-way ANOVA with repeated measures to determine the effects of treatment over time. Fifty percent withdrawal thresholds to mechanical stimuli were compared with nonparametric Kruskal–Wallis or Mann–Whitney U test. Data represent the mean ± SD. Values below the 5% probability level were considered significant.
Construction and functional characterization of adenoviral vector
To examine the ability of Sema3A to reduce NGF-induced sprouting within the spinal cord, we generated Sema3A/Ad. To verify biological activity of virus-derived Sema3A, U373 cells were transfected using 30 pfu/cell GFP/Ad (negative control) or Sema3A/Ad. This astrocyte cell line was chosen because of its similar transduction efficiency and cellular properties to primary astrocytes. Aggregates of transfected cells were cocultured with E10 chicken DRG in three-dimensional collagen gels supplemented with NGF to enhance survival of the sensory neurons (Ruit et al., 1992; Zhang et al., 1994). DRG cultured with Sema3A/Ad transfected aggregates demonstrated a strong chemorepulsive response, in which axons grew in a direction opposite to the transfected cell aggregate (Fig. 1B), whereas DRG cultured with GFP-expressing U373 cells showed a more even, circular growth pattern (Fig. 1A). Interestingly, in all cultures, axons appeared to avoid direct contact with the U373 cells, suggesting contact-mediated inhibition between these axons and U373 cells. This inhibitory effect was attributed to U373 cells themselves, because a statistically identical growth pattern was observed with untransfected U373 (220 ± 20°). To quantify the extent of chemorepulsion, the circumferential degree of axonal growth was measured in relationship to the target cell aggregate (Fig. 1C). DRG cultured with GFP/Ad-transfected aggregates displayed a growth angle of 223 ± 17°, whereas a much smaller axonal growth angle (93 ± 38 °) in a direction opposite to the Sema3A-secreting cells was observed (Fig. 1D).
Sema3A repulsion is regulated by NGFin vitro
To investigate the extent by which NGF influenced Sema3A chemorepulsion if expressed by a single source, U373 cells were cotransfected with 30 pfu/cell Sema3A/Ad and different concentrations of NGF/Ad. Western blot confirmed consistent secretion of Sema3A (∼97.4 kD) and dose-dependent increases in NGF (∼14 kD) production with increased NGF virus titers (Fig. 2A). At the highest titer of NGF/Ad (30 pfu/cell), which was equivalent to the Sema3A/Ad titer, similar levels of protein were expressed for Sema3A and NGF. Exact quantification could not be done because of lack of an ELISA assay for Sema3A. Cotransfection of U373 with Sema3A/Ad and NGF/Ad established a competitive gradient that was used to examine axonal growth in the presence of a dose-dependent increase in NGF chemoattraction against a constant expression level of Sema3A. The growth pattern of DRG cultured with aggregates cotransfected with Sema3A and lower titers of NGF/Ad (Fig. 2D–F) appeared to be similar to that with aggregates transfected with Sema3A/Ad alone (Fig. 2C). At the highest NGF concentration, Sema3A failed to induce repulsion, resulting in growth patterns that were indistinguishable from GFP controls (Fig. 2G). DRG cocultured with explants treated with NGF/Ad alone showed little to no repulsion (Fig. 2H). Measurements of DRG growth angles (Fig. 2B) confirmed the NGF dose-dependent abolishment of Sema3A-induced repulsion (F(6,60) = 33.37; p < 0.0001).
Sema3A inhibits NGF-induced sprouting of CGRP-positive sensory afferents in adult rat spinal cord in a dose-dependent manner
Application of NGF within the spinal cord has been shown to induce robust sprouting of CGRP-positive sensory axons (Christensen and Hulsebosch, 1997; Romero et al., 2000, 2001; Gwak et al., 2003); however, the possibility remains that increased CGRP staining could be attributable to a phenotypic switch in neuropeptide expression within a myelinated sensory subpopulation (Neumann et al., 1996). To examine this possibility, sciatic nerves of NGF/Ad-treated animals were injected with CTB, which is transported transganglionically in large- and medium-diameter myelinated sensory axons. For these experiments, CTB was injected bilaterally and showed equivalent staining between the control and NGF/Ad-injected sides (Fig. 3A, brown staining). The density of CGRP-positive axons on the NGF/ Ad-injected side, however, increased dramatically (Fig. 3A, blue staining). Higher magnification revealed that the CTB and CGRP are localized to separate axonal populations and, at best, only very few axons were labeled for both markers (Fig. 3B,C). This was further confirmed using immunofluorescence that clearly shows separation of the two axon populations (Fig. 3D,E). To further confirm that NGF/Ad injections did not alter the distribution of other nociceptive axonal subpopulations, sections were also double-labeled for IB4 and CGRP (Fig. 3F). IB4 labels a glial cell line-derived neurotrophic factor-responsive subpopulation of nociceptive axons that are localized to the inner region of lamina II. Overexpression of NGF caused no observable change in the sprouting or distribution of these axons (Fig. 3F). These data demonstrate that overexpression of NGF within the spinal cord directly increase sprouting of CGRP-positive axons without altering neuropeptide expression or sprouting of the other sensory axon subpopulations examined.
To test the hypothesis that Sema3A could inhibit NGF-induced sprouting of nociceptive fibers, Sema3A/Ad with different titers of NGF/Ad were coinjected into the right dorsal horn of adult rat spinal cords. Sema3A and NGF expression was examined 10 d after unilateral injections of Sema3A/Ad combined with NGF/Ad at titer ratios of 1:0.04, 1:0.1, 1:0.2, or 1:1. Western blot analysis shows Sema3A expression within the spinal cord of animals coinjected with the highest titer of NGF/Ad (Fig. 4A). NGF protein concentrations within spinal cords were determined by ELISA and reflected a titer-dependent dose–response of NGF expression (Fig. 4B). All concentrations were well above endogenous levels, which vary between 0.4 and 1.3 ng/mg spinal cord tissue with this ELISA assay.
CGRP is a specific marker of small-diameter sensory afferents that terminate primarily in laminas I and II of the dorsal spinal cord (Gibson et al., 1984; Chung et al., 1988), and NGF induces robust sprouting of these afferents into deeper dorsal horn laminas (Oudega and Hagg, 1996; Romero et al., 2000, 2001). The extent of CGRP-positive axon sprouting was compared between spinal cords coinjected with either Sema3A/Ad or GFP/Ad and NGF/Ad at either a 1:0.04 or 1:0.2 ratio. These ratios were chosen because, in pilot experiments, no inhibition of NGF-mediated sprouting was observed at equal titers of Sema3A/Ad and NGF/Ad. In addition, these titer ratios demonstrated effective Sema3A chemorepulsion in vitro. Consistent with our previous study, extensive sprouting of CGRP-positive fibers throughout the ipsilateral spinal cord, including the intermediate zone, ventral horn, and lateral funiculus, was observed 2 weeks after coinjection of GFP/Ad with NGF/Ad at either concentration (Fig. 4D,F). High magnification at the lateral border of the intermediate zone, where very few sensory axons would normally appear, demonstrates the extent of CGRP-positive axonal sprouting (Fig. 4D–G). Numerous CGRP-positive axons were also observed growing along blood vessels throughout this region and the dorsal horn. Coinjection of Sema3A/Ad with low concentration of NGF/Ad (1:0.04) exhibited a greater inhibition on CGRP-positive fibers (Fig. 4G) compared with higher concentrations of NGF/Ad (Fig. 4E). At higher NGF concentrations, CGRP-positive axons sprouted throughout the entire ipsilateral spinal cord, albeit at lower densities (Fig. 4E), even into regions expressing Sema3A, as determined in double-labeled sections (data not shown). To quantify the extent of CGRP fiber sprouting between the treatment groups, the area occupied by these fibers was measured in the right dorsal quadrant of the cord. Sema3A reduced NGF-mediated sprouting of CGRP-positive fibers at both concentrations, but statistically significant inhibition (F(3,20) = 5.24; p < 0.01) by Sema3A was only achieved at the lower concentration of NGF (Fig. 4C). In the presence of low concentrations of NGF, Sema3A did not appear to affect the density of endogenous CGRP-positive axons within laminas I and II compared with the contralateral side.
Characterization of the nociceptive fibers inhibited by exogenous Sema3A
Our previous studies show that NGF/Ad administration selectively induces neurite sprouting of CGRP and substance P (SP)-containing fibers, without affecting the non-peptidergic IB4-positive fibers (Fig. 3F) (Romero et al., 2000). To further characterize the effect of Sema3A on subgroups of nociceptive afferents, we also examined inhibition of SP-positive fiber sprouting. Consistent with previous experiments, SP-positive fibers extended neurites in response to injection of NGF/Ad (5 × 104 pfu/μl), in a pattern resembling CGRP fiber sprouting, albeit at a much lower fiber density (Fig. 5A,B). Similar to CGRP-positive fibers, no sprouting of SP-positive fibers was observed when Sema3A/Ad was coinjected with NGF/Ad (Fig. 5C,D). Measurements also confirm a significant reduction in the SP fiber-occupying area after Sema3A/Ad injection compared with control (GFP/Ad and NGF/Ad) groups (Fig. 5E).
Sema3A overexpression attenuates the NGF-induced pain responses
In a previous study, we observed that NGF-induced hyperinnervation of CGRP- and SP-positive fibers at the fourth and fifth lumbar segments correlated with the appearance of chronic pain and hyperalgesia (Romero et al., 2000). To identify whether Sema3A chemorepulsion of these nociceptive afferents could reduce NGF-mediated pain responses, tests of thermal and mechanical nociception were used. Measurements of PWL to thermal stimuli revealed the appearance of thermal hyperalgesia 2 weeks after coinjection of GFP/Ad and NGF/Ad. With coexpression of Sema3A, a lower level of hyperalgesia was observed (Fig. 6B), but the difference was not statistically significant (p < 0.1). To examine mechanical allodynia, von Frey hairs were used to calculate the 50% paw withdrawal threshold. Two weeks after coinjection of GFP/Ad and NGF/Ad, a significant decrease in the 50% withdrawal threshold was observed compared with preinjection or contralateral hindpaw thresholds. Coinjection with Sema3A/Ad, however, significantly increased the threshold toward preinjection or contralateral levels (p < 0.01) (Fig. 6D). Another characteristic of chronic pain is a guarding behavior and spontaneous or touch-mediated vocalizations. These behaviors were still observed in Sema3A-treated animals, although the extent of these behaviors seemed reduced. Taken together, these data suggest that Sema3A inhibition of CGRP- and SP-positive fiber sprouting is able to reduce some NGF-induced pain responses but fails to fully prevent the progression of neuropathic pain.
Previously, we demonstrated that Sema3A exerts a repulsive effect on trigeminal sensory nerve terminals innervating the adult rabbit cornea in vivo (Tanelian et al., 1997). However, to date, only indirect evidence exists for a functional role for Sema3A in shaping sensory axon plasticity within the adult spinal cord. NGF-responsive neurons in adult DRG express Sema3A receptor protein neuropilin-1 (Gavazzi et al., 2000) and collapse in response to Sema3A treatment in vitro (Reza et al., 1999). In this study, we demonstrate that Sema3A retains its chemorepulsive effect on NGF-dependent axons within the adult spinal cord in vivo and that this molecule can be used to limit NGF-induced sprouting of nociceptive axons, resulting in a partial reduction in neuropathic pain.
Continual expression of neuropilin-1 by sensory neurons and Sema3A by motor neurons (Pasterkamp et al., 1998) suggest that the receptor functions to restrict sensory afferent plasticity and prevent sprouting in the normal adult spinal cord (Gavazzi, 2001). According to this hypothesis, alterations in signaling, in this case mediated by NGF, that increase the growth response within nerve terminals above the Sema3A chemorepulsive threshold would initiate the formation of a growth cone and, thus, induce growth or sprouting. This would have the overall effect of increasing the size of the terminal branches. The addition of exogenous Sema3A would increase the occupancy of neuropilin-1 receptor complexes and, thus, readjust the signaling threshold within the nerve terminal to induce growth cone collapse and prevent sprouting. Our data support this hypothesis, because Sema3A inhibited sprouting at the lower, more physiological concentrations of NGF examined.
The expression levels for NGF in our experiments ranged from 4- to 30-fold above endogenous spinal cord levels. At very high NGF concentrations, Sema3A was incapable of inducing chemorepulsion in vitro or inhibiting axonal sprouting in vivo. Our in vivo data agree with the in vitro observation that neurotrophins modulate Sema3A-mediated growth cone collapse (Song et al., 1998; Tuttle and O'Leary, 1998; Dontchev and Letourneau, 2002). These data are particularly supportive of recent observations by Dontchev and Letourneau (2002), which demonstrated a NGF dose-dependent reduction of growth cone collapse. Strong evidence indicates that neurite growth and growth cone mobility are modulated by the Rho family of small GTPases (Dickson, 2001; Song and Poo, 2001). Activation of TrkA receptors by NGF increases cyclic nucleotides and activates cAMP-dependent protein kinase A (PKA), which is thought to reduce myosin contraction mediated by RhoA GTPase (Cai et al., 1999; Dickson, 2001; Dontchev and Letourneau, 2002). Increased levels of cyclic nucleotides within the growth cone prevent collapse and transform the chemorepulsive signal mediated by Sema3A to attraction (Song et al., 1998; Song and Poo, 2001; Dontchev and Letourneau, 2002) and, thus, override Sema3A inhibition of sprouting.
During development, NGF and Sema3A are expressed within the spinal cord and influence pathway guidance of nociceptive axons. Under such circumstances, growth cone guidance is influenced by the coincidental detection of these, and possibly other, signals resulting in the termination of growth within upper dorsal horn. The ability of high concentrations of NGF to reduce the chemorepulsive efficacy of Sema3A might provide a potential mechanism to explain injury-induced sprouting of these axons. Injury to the adult spinal cord induces an upregulation of both NGF and Sema3A; however, these molecules are localized to different compartments. Semaphorin expression is restricted to fibroblasts within the wound and ventral motor neurons (Pasterkamp et al., 1998, 2001; De Winter et al., 2002), whereas NGF is produced by inflammatory cells and astrocytes throughout and surrounding the lesion site (Krenz and Weaver, 2000; Nakamura and Bregman, 2001). This expression pattern would likely generate higher concentrations of NGF than Sema3A in the dorsal spinal cord and induce axonal sprouting. Several lines of evidence support this mechanism: (1) Sema3A-mediated inhibition of adult nociceptive axon sprouting is dependent on the concentration of NGF in vivo; (2) preconditioning neurons with NGF reduces Sema3A-mediated growth cone collapse in vitro (Dontchev and Letourneau, 2002); (3) NGF increases cyclic nucleotides and activates cAMP-dependent PKA, which reduces myelin and Sema3A-mediated growth cone collapse (Song et al., 1998; Cai et al., 1999; Dontchev and Letourneau, 2002); and (4) anti-NGF treatment reduces sprouting of these axons (Christensen and Hulsebosch, 1997; Krenz and Weaver, 2000). Growth cone behavior and sprouting of adult nociceptive axons are, therefore, modulated by coincident signaling between neurotrophins and semaphorins and depend on a balance between these guidance molecules (Dontchev and Letourneau, 2002). However, at the upper limits of expression of both Sema3A and NGF, our data suggest that NGF-mediated signaling overwhelms Sema3A-mediated repulsion and axon sprouting occurs. The reasons for this effect are unknown but may be attributable to reduced responsiveness to Sema3A because of differences in receptor density on the axon (Owesson et al., 2000), or reduced presence of the cell adhesion molecule L1, which is required for Sema3A/neuropilin-1 signaling (Castellani et al., 2000; Romero et al., 2001). The latter is particularly noteworthy, because we have observed identical inhibition of NGF-induced sprouting by the overexpression of L1 but not neural cell adhesion molecule or N-cadherin (Romero et al., 2001).
Chronic pain and autonomic dysreflexia
Upregulation of NGF, as seen after peripheral tissue injury, peripheral nerve injury, and spinal cord injury, has been shown to be associated with the development of both acute and chronic pain and can be reversed by administration of anti-NGF antibodies (Woolf et al., 1994; McMahon et al., 1995; Christensen and Hulsebosch, 1997). In the corneal model of peripheral nociception, tissue injury is known to increase NGF (Lambiase et al., 2000), resulting in increased peripheral nerve terminal density and electrophysiological nociceptor responsiveness (Tanelian and Monroe, 1995). In the corneal lesion model, increased Sema3A expression significantly reduced corneal nociceptive axon sprouting and nerve terminal density at endogenous NGF concentrations (Tanelian et al., 1997). Our more recent in vivo studies on spinal cord found upregulation of NGF to induce aberrant sprouting of nociceptive fibers throughout the dorsal horn, accompanied by hyperalgesia and guarding behavior in rats (Romero et al., 2000). Two potential mechanisms by which NGF induces hypersensitivity to pain are by increasing the sprouting of neuropeptide-containing nociceptors and the release of CGRP, SP, or BDNF as neuromodulators of pain (Woolf, 1996; Christensen and Hulsebosch, 1997; Pezet et al., 2002; Gwak et al., 2003). Sema3A-mediated reductions in mechanical allodynia and thermal hyperalgesia are attributed to reductions in the terminal field density associated with sprouting of these nociceptive axons. In our studies, the reversal of hyperalgesia was not complete, suggesting that other mechanisms besides NGF-induced hyperinnervation may participate in the altered pain response. One such possibility is the continual increased production and release of CGRP, SP, or BDNF at normal sites within laminas I and II. NGF has been shown to directly regulate CGRP expression via the cAMP and p42/p44 mitogen-activated protein kinase pathways (Freeland et al., 2000), which have not been shown to be influenced by signaling through the neuropilin-1/plexin receptor (Pasterkamp and Kolodkin, 2003). Therefore, semaphorin overexpression should not affect the function of previously established synapses that play a major role in the progression of neuropathic pain. For example, continual expression of neuropeptides or BDNF could act to increase the "gain" of these synapses and reduce the pain threshold (Malcangio et al., 1997; Thompson et al., 1999). Alternatively, rearrangement of non-NGF-responsive terminals may alter central circuits to change innocuous stimulation into noxious sensation (Woolf et al., 1995), which we observed as touch-mediated vocalization in NGF/Sema3A-treated animals.
Another manifestation of increased NGF expression after spinal cord injury is the development of autonomic dysreflexia attributable to aberrant sprouting of NGF-responsive, CGRP-immunoreactive neurons (Jacob et al., 2001; Weaver et al., 2001). Neutralizing intraspinal NGF was able to block autonomic dysreflexia caused by spinal cord injury (Krenz et al., 1999). We are presently examining whether upregulation of Sema3A in the adult spinal cord will be able to prevent or reverse autonomic dysreflexia after spinal cord injury. The findings here lay the groundwork for using Sema3A therapeutically to reduce the aberrant sensory neurite growth that occurs when NGF levels are increased in the adult spinal cord and can lead to acute or chronic pain and autonomic dysreflexia.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS 38126. We thank Melody King, Raymond Collins, and Lorna Putnam for histological assistance; Udesh DeSilva for quantification of outgrowth; and Dr. Diane M. Snow and Jeffrey D. Smith for providing fertilized chick eggs.
Correspondence should be addressed to Dr. George M. Smith, Department of Physiology, MS 508, Spinal Cord and Brain Injury Research Center, University of Kentucky Chandler Medical Center, Lexington, KY 40536-0298. E-mail: gmsmith{at}uky.edu.
Copyright © 2004 Society for Neuroscience 0270-6474/04/240819-09$15.00/0


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