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TRPV1 mediates astrocyte activation and interleukin-1β release induced by hypoxic ischemia (HI)
Journal of Neuroinflammation volume 16, Article number: 114 (2019)
Hypoxic-ischemic encephalopathy (HIE) is a serious birth complication with high incidence in both advanced and developing countries. Children surviving from HIE often have severe long-term sequela including cerebral palsy, epilepsy, and cognitive disabilities. The severity of HIE in infants is tightly associated with increased IL-1β expression and astrocyte activation which was regulated by transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel in the TRP family.
Neonatal hypoxic ischemia (HI) and oxygen-glucose deprivation (OGD) were used to simulate HIE in vivo and in vitro. Primarily cultured astrocytes were used for investigating the expression of glial fibrillary acidic protein (GFAP), IL-1β, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and activation of the nucleotide-binding, oligomerization domain (NOD)-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome by using Western blot, q-PCR, and immunofluorescence. Brain atrophy, infarct size, and neurobehavioral disorders were evaluated by Nissl staining, 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining and neurobehavioral tests (geotaxis reflex, cliff aversion reaction, and grip test) individually.
Astrocytes were overactivated after neonatal HI and OGD challenge. The number of activated astrocytes, the expression level of IL-1β, brain atrophy, and shrinking infarct size were all downregulated in TRPV1 KO mice. TRPV1 deficiency in astrocytes attenuated the expression of GFAP and IL-1β by reducing phosphorylation of JAK2 and STAT3. Meanwhile, IL-1β release was significantly reduced in TRPV1 deficiency astrocytes by inhibiting activation of NLRP3 inflammasome. Additionally, neonatal HI-induced neurobehavioral disorders were significantly improved in the TRPV1 KO mice.
TRPV1 promotes activation of astrocytes and release of astrocyte-derived IL-1β mainly via JAK2-STAT3 signaling and activation of the NLRP3 inflammasome. Our findings provide mechanistic insights into TRPV1-mediated brain damage and neurobehavioral disorders caused by neonatal HI and potentially identify astrocytic TRPV1 as a novel therapeutic target for treating HIE in the subacute stages (24 h).
HIE resulting from oxygen deprivation and blood flow reduction to the brain is a common cause of brain damage [1]. The mechanisms involved in the brain damage include excitotoxicity, apoptosis, and glial overactivation [2]. Although the importance of microglial activation in hypoxia-induced neuroinflammation is well explored [3], the role of astrocyte activation in this process has attracted more attention. It has been explored that activation of astrocytes under pathological conditions [4], such as ischemic damage [5] and neuroinflammation [6,7,8], are closely related to the pathogenesis of brain injury [9]. Activation of astrocytes and subsequent increase of IL-1β release are shown to be a key determinant of the outcome and prognosis of HIE [10,11,12]. Astrocytes are the predominant source of IL-1β in post-traumatic stress disorder [13] and prion disease [14]. Active NLRP3 inflammasome, which cleaves the pro-inflammatory cytokines IL-1β to its active form [15], has been detected in astrocytes in several animal models of intracerebral hemorrhage, such as ischemia/reperfusion injury and hemorrhage transgenic amyotrophic lateral sclerosis [16,17,18,19]. Moreover, inhibiting the activation of NLRP3 inflammasome attenuates neonatal HI-induced brain injury in rats [20, 21], and systemic neutralizing IL-1β by infusions of anti-interleukin-1β antibodies reduces short-term brain injury after cerebral ischemia in the ovine fetus [22].
TRPV1 channel is the prototypic member of the TRP family and displays dynamic ion selectivity for ions including H+, Na+, Ca2+, and Mg2+ [23]. In the CNS, TRPV1 participates in synaptic transmission, neurogenesis, and neuroinflammation [24, 25]. Recent studies suggest that TRPV1 is functionally expressed in CNS glial cells, especially microglia and astrocytes [26, 27]. In astrocytes, TRPV1 activation mediates its migration, chemotaxis, and the expression of GFAP, during stress and injury [28]. TRPV1 activation stimulates a JAK2-STAT3 pathway to regulate astrocyte activation, astrocyte hypertrophy, and the expression of cytokines such as IL-1β and IL-6 in vitro and in vivo [29,30,31,32]. TRPV1 activation participated in neonatal brain injuries [33, 34], and overactivated astrocytes exacerbate neurological deficits [35]. These findings strongly suggest that TRPV1 may mediate astrocytic activation and IL-1β release in HIE.
We have previously reported that TRPV1 promoted the activation of microglia and upregulated the expression of TNF, IL-1β, IL-6, and high mobility group box 1 (HMGB1) in repetitive febrile seizure (FS) mice [36]. The main purpose of the present study was to determine whether TRPV1 aggravated brain damages, neurological deficits by promoting astrocytes activation, and neuroinflammation in a mouse model of neonatal HI. We found that in the subacute stage (24 h), TRPV1 activation increases the phosphorylation of JAK2 and STAT3, which results in an increased expression of GFAP and IL-1β. TRPV1 activation promotes the activation of the NLRP3 inflammasome, which drives the release of IL-1β. Furthermore, knocking out TRPV1 significantly improves neonatal HI-induced neurobehavioral disorders.
Materials and methods
Animal models and treatments
B6.129X1-TRPV1 KO mice used in experiments were from the Jackson Laboratory, and wild-type (WT) mice were provided by the Hubei Province Center for Animal Experiments. Animal experiments were approved by the Care and Use Committee of Wuhan University Medical School. B6.129X1-TRPV1 KO mice and wild-type mice were both on a C57BL/6J background. Mice were grouped randomly and kept on a 12-h light–dark cycle, in a room maintained at 25 ± 2 °C and relative humidity of 60~80%. All mice were housed at the animal facility of the Animal Biosafety Level III Laboratory (ABSL-III) of the Wuhan University. The day of the birth was defined as postnatal day zero (P0). Mice pups (n = 160) of P9 were used in this study and randomly divided into the five groups (n = 32 each group): WT-Sham, WT-HI, WT-Capsaicin-HI (intraperitoneal injection of 3 mg/kg capsaicin 0.5 h before neonatal HI), KO-Sham, and KO-HI.
Neonatal hypoxia-ischemia model
A well-characterized model of neonatal HI was followed as a previously described [2, 37]. P9 C57BL/6 WT and TRPV1 KO mice of both genders (5 ± 1 g of body weight, equal males, and females were chosen for each group) were anesthetized by inhalation of isoflurane. The sterilized skin was incised with an ophthalmology scissor. And the right pulsating carotid artery was carefully separated. The upper and lower ends of the right carotid artery were tied using 4-0 surgical suture before cutting the artery in the middle. The skin incision was sutured with the same surgical suture. All the above experimental surgical instruments were sterilized. After 2 h of recovery, the pups were placed in an airtight transparent chamber, and the chamber was placed into a 37 °C incubator to maintain a constant thermal environment. The pups were maintained in 8% O2 in N2 for 45 min. After the hypoxic process, the mouse pups were put back in the cages. Successful HI model showed significant edema in the ipsilateral hemisphere, while the sham group (underwent anesthesia, neck incision, and suture) did not. The mortality of this model was about 10%.
Nissl staining
We followed detailed protocols that have been previously reported [38]. Briefly, at 24 h after neonatal HI, mice pups were perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA). The brains were then obtained for Nissl staining following the standard protocol.
Infarct volume measurement
The procedure has been previously described [39]. At 24 h post-HI, animals were transcardially perfused with PBS under deep anesthesia. The brains were obtained and sectioned into 2 mm slices and then immersed in 2% 2, 3, 5-triphenyltetrazolium chloride monohydrate (TTC) solution at 37 °C for 10 min, followed by 4% PFA to completely fix the tissue. The infarct volume was traced and analyzed by ImageJ software (version 1.41).
Primary mouse cortical astrocytes cultures
Astrocyte culture was prepared as previously described [40]. P0 mice were euthanized after being disinfected with 75% ethanol. The brain tissue was isolated and then put into the pre-cooled PBS. In order to obtain dissociated cells, meninges were removed and the clean cerebral cortex was digested in Hank's balanced salt solution (HBSS) containing 0.125% trypsin at 37 °C for 10 min. Complete growth media (1× Dulbecco's modified Eagle's medium (DMEM)/F12, 10% heat-inactivated fetal bovine serum, 1% l-glutamine, and 1% penicillin/streptomycin) were added to terminate the digestion. Gently blow digested tissues with pap dropper and then centrifuge at 1000 rpm for 5 min. Dissociated cells were planted in T75 flasks in glial medium and then maintain at conventional cell culture incubator (37 °C, 5% CO2) for 10–12 days. To purify astrocytes from microglia and oligodendrocytes, the cells in the T75 were subjected to continuous shaking at 37 °C (200 rpm, 10 h). The remaining cells were identified by immunofluorescence staining with anti-GFAP antibody, and approximately 95% of the remaining cells were GFAP-positive (data not shown).
OGD progression
Oxygen-glucose deprivation (OGD) was conducted as described previously [41]. Briefly, astrocytes were grown in complete growth media for 9 days as monolayers in cell culture incubator (95% O2 and 5% CO2 at 37 °C). And all groups of cell medium were changed to serum-free growth medium 24 h before OGD. To initiate OGD in vitro, the plates were washed with PBS three times, added OGD medium (serum- and glucose-free DMEM), and placed in a hypoxic/anoxic chamber (1% O2, 5% CO2, and 94% N2 at 37 °C); 3.5 h later, plates were removed from the anaerobic chamber and OGD medium was changed to complete growth medium. At the same time, control glucose-containing cultures remained in a regular incubator (5% CO2 and 95% O2) and the medium was changed to fresh complete growth medium with OGD group. The supernatants and cell extracts were collected after OGD for the following experiments.
Real-time PCR
Total RNA was extracted from the cortical-derived astrocytes of WT and TRPV1 KO mice from different groups using TRIzol reagent (Invitrogen Life Technologies Corporation, USA) to detect the mRNA level of IL-1β, IL-6, TNF, IL-10, NLRP3, an apoptosis-associated speck-like protein containing carboxyl-terminal CARD (ASC), and caspase-1.
The quantity of total RNA was measured by a UV spectrophotometer (Biochrom Ltd., UK). Next, reverse transcription was performed using a cDNA synthesis kit (TaKaRa Biotechnology). QPCR was performed on a SYBR-Green premix (TransGen Biotech) according to the manufacturer's specification with the primers listed in Table 1 (Sangon Biotech, Shanghai, Co., Ltd.). The cycling parameters for the CFX96 sequence detection system were 95 °C for 5 min; 40 cycles of 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 20 s. The expression of target genes was normalized to the mRNA level of β-actin as an internal control. The ΔΔCt values of each group were analyzed, and the mRNA expression of different groups was normalized to 2−ΔΔCt.
Western blot analysis
Western blotting was performed according to the manufacturer's specification. The brain tissue samples and astrocytes were collected after neonatal HI and OGD. Proteins were extracted by homogenizing in RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with phenylmethanesulfonyl fluoride (PMSF) and further centrifuged at 12,000 rpm at 4 °C for 10 min. The protein concentrations were measured using a detergent-compatible assay (Bio-Rad, Dc protein assay). Equal amounts of protein were loaded on an SDS-PAGE gel. After electrophoresis and transfer to a polyvinylidene fluoride (PVDF) membrane, the membranes were blocked by 5% skimmed milk for 3 h. The membranes were incubated with the GFAP, JAK2, and STAT3 antibody overnight at 4 °C. Details for the primary and secondary antibodies are listed in Table 2. After incubation, the membranes were washed at least three times with TBST (TBS containing 0.2% Tween-20) and were then incubated for 1.5 h with secondary antibodies at room temperature. The membranes were washed again with TBST three times. Finally, the reaction was developed using a chemiluminescent reagent (ECL; Ecl Advantage Inc., Menlo Park, California, USA) and the bands of different proteins were detected using an imaging densitometer (Bio-Rad, Foster City, CA, USA). The data were analyzed using ImageJ software (version 1.41).
Measurement of cytokine by enzyme-linked immunosorbent assay (ELISA)
The amounts of IL-1β in the culture supernatants in each group of astrocytes were measured by ELISA kit (purchased from 4A Biotech Co., Ltd) according to the manufacturer's instructions. The serum samples of animals or cells from different groups were added into a 96-well plate. Then, it was incubated with the corresponding primary antibody followed for 90 min at 37 °C. After washing three times with wash buffer, the secondary antibody was added into the plate and incubated for 60 min at 37 °C. And then the absorbance at 450 nm (Thermo, USA) was recorded. The concentration of the target protein was calculated according to the standard curve and normalized to the total protein of the samples. Assays were performed independently three times using triplicate wells.
Cell viability assay
Cell counting kit-8 (CCK-8) assay was performed as previously described [42]. Astrocytes were seeded at a density of 3 × 104 mL−1 in 96-well plates. Cell viability was subsequently assessed using the CCK-8 (Dojindo, Tokyo, Japan) according to the manufacturer's instructions. The optical absorbance at 450 nm was detected using a plate reader (Thermo Fisher Scientific).
Immunofluorescence staining was used to detect GFAP and IL-1β expression in peri-ischemic brain tissue, assembly of ASC speck and NLRP3 and caspase-1 expression in astrocytes.
Mice in each group were deeply anesthetized with 10% chloral hydrate and transcardially perfused first with PBS and then fixed in 4% paraformaldehyde solution at room temperature, dehydrated 24 h after HI. Serial coronal sections (5-μm thick with injury epicenter located centrally) prepared with cryotome (Leica, Wetzlar, Germany) were used for immunofluorescence labeling. Sections were incubated with 5% BSA for 30 min at 37 °C. The tissue slices were then incubated overnight with the anti-GFAP and anti-IL-1β antibodies. For the assessment of assembly of ASC speck and NLRP3 and caspase-1 expression in astrocytes, cells were fixed with methanol, washed with PBS three times, and incubated at 4 °C with anti-ASC, anti-NLRP3, and anti-caspase-1 antibodies. On the following day, the sections and cells were washed and subsequently incubated with secondary antibodies Cy3-conjugated Goat Anti-Rabbit IgG (H + L) and Alexa Fluor® 488 Conjugates Goat Anti-Mouse for 1 h at 37 °C. Dilutions for antibodies were listed in Table 3. Images obtained using a confocal microscope (Leica-LCS-SP8-STED). And images area and intensity were measured in ImageJ.
Morphological assessment
The morphological assessment of astrocytes after OGD was performed according to M. Neal [43]. Briefly, 20 × images (Olympus U-HGLGPS) were imported into ImageJ for morphological analysis. The glial processes could be analyzed using an ImageJ plugin named Analyze Skeleton [44]. The image was converted to binary and then skeletonized in ImageJ software, and then, the skeletonized image was put into the Analyze Skeleton plugin to get detailed information about the branch length of each cell. Cell soma size was measured by freehand selection outlining each cell soma and then selecting the "area" analyzes to measure. At least three fields were used for each section, and then, three biological replicates were used for each experiment. At least 20 cells were measured for each of the morphology assessments.
Neurobehavioral testing
Cliff aversion reaction, Geotaxis reflex and Grip test were respectively assessed at 1 day (P10), 3 days (P12), and 7 days (P16) after neonatal HI. All of the above experiments were observed by at least two uninformed laboratory workers, and all groups of animals maintained the same test method.
Cliff aversion reaction was used to assess the ability of rodents' respond to adverse environments [45]. Put two planks together and the board was 30 cm above the ground. The head and front upper limbs of the mouse were on the first plank, the rest of the body was on the second plank, and all the mice were placed in this position. When the mouse relaxed, the first plank was quickly lowered so that the bilateral forelimbs of the mouse were suddenly suspended. After a short delay, the mouse can return to the second plank. The time required for the mouse to withdraw the forelimb was examined. Each mouse was tested three times. If the forelimbs were not withdrawn for more than 60 s, it would be recorded as 60 s. Grip test was used to assess the performance and tolerance of mice [46]. The bilateral forelimbs of the mice were grasped on a metal wire (1.5 mm in diameter), the metal rope was 50 cm from the ground, and the ground was covered with cork. Record the time from the mouse holding the metal rope to releasing the metal rope and test each mouse three times.
Geotaxis reflex was considered to diagnose the function of vestibular and proprioception of mice [47]. A wooden board was placed at an angle of 45°, and then, the mouse was placed at the middle of the wood heading toward the lower end of the wood. The time required for the mouse to turn 180° toward the upper end of the board was recorded. One mouse was tested three times. If the time was more than 60 s, it would be regarded as the maximum value of 60 s.
Experimental design and statistical analysis
Prism 7 (RRID: SCR_002798) software was used to analyze data and form the graphs in this work (including which tests were performed, exact P values, and sample sizes). Simply, one-way ANOVA with a test for linear trend, Tukey test was used as appropriate to analyze parametric statistics. At least three independent experiments were applied to collect effective data. Bias was avoided by making sure that assessor was blinded to collecting and analyzing data. P < 0.05 was considered significant. Average values represent the mean ± SEM. In the quantitative analysis of Fig. 5e–g, the number of animals used in neurobehavioral testing was indicated in figure legends. *P represents the significance compared to sham or control. #P represents the significance compared to WT-HI or WT-OGD.
TRPV1 deficiency prevents the upregulation of GFAP expression induced by neonatal HI
To determine whether TRPV1 mediates astrocyte activation following neonatal HI insult, the expression of GFAP were investigated after neonatal HI in both TRPV1 KO mice and congenic WT C57BL/6 mice. Immunofluorescence staining revealed significantly stronger labeling of GFAP in the ipsilateral hippocampus at 12 h, 24 h, and 48 h after neonatal HI compared to the sham group (Fig. 1a and Additional file 1: Figure S1A), and its expression saturated at 24 h (Fig. 1b, c). The labeling of Iba1 increased at 6 h, 12 h, and 24 h (Additional file 1: Figure S1A). Western blot results (Fig. 1d, e) showed that GFAP protein level was gradually upregulated at 12 h, 24 h, and 48 h after neonatal HI compared with the sham group, suggesting that HI insult significantly increases the number of activated astrocytes in the hippocampus.
On the other hand, immunofluorescence (Fig. 1f–h) and Western blot results (Fig. 1i, j) showed that the neonatal HI-induced increase of GFAP expression was significantly reduced at 24 h in the TRPV1 KO mice compared with WT mice. Consistent with the genetic ablation result, systemic desensitization of TRPV1 (intraperitoneal injection of 3 mg/kg capsaicin 0.5 h before neonatal HI) significantly reduced the increase of GFAP expression caused by neonatal HI in WT mice [48,49,50]. Knocking out TRPV1 mildly reduced Iba1+ cell, while intensely reduced GFAP+ cell in the ipsilateral hemisphere hippocampus (Additional file 1: Figure S1). Taken together, these results suggest that either genetic ablation of TRPV1 or systemic desensitization of TRPV1 effectively suppress the upregulation of GFAP expression induced by neonatal HI, highlighting the importance of TRPV1 in neonatal HI-induced astrocyte activation.
Activation of astrocytes and increased expression of inflammatory cytokines induced by OGD are TRPV1-dependent
To explore the role of TRPV1 on HI-induced activation of astrocytes in vitro, hippocampal astrocytes were isolated and cultured to undergo OGD. Either TRPV1 deficiency or capsaicin-induced TRPV1 desensitization, pretreating with capsaicin (10 μM) 0.5 h before OGD, prevented astrocytes from cellular edema (Fig. 2a, b), reduction of branch processes (Fig. 2a, c), and cell death (Fig. 2d) at 24 h after OGD. In addition, the upregulation of GFAP reduced in TRPV1 deficiency astrocytes and WT capsaicin pretreatment astrocytes (Fig. 2e, f).
Activated astrocytes release several cytokines in neurological diseases [9]. OGD challenge significantly increased IL-1β (Fig. 2g), IL-6 (Fig. 2h), and TNF (Fig. 2i) mRNA expression level, which was inhibited by genetic ablation and capsaicin-induced desensitization of TRPV1 (Fig. 2g–i). Of note, although the expression level of the anti-inflammatory cytokine (IL-10) increased after OGD, genetic ablation and capsaicin-induced desensitization of TRPV1 only slightly inhibited the upregulation (Fig. 2j). These results suggested that TRPV1 is required for astrocyte activation, morphological change, and the expression of pro-inflammatory cytokines, especially IL-1β and IL-6 in vitro.
Activation of JAK2-STAT3 signaling in astrocytes is required for OGD-induced TRPV1-dependent expression of IL-1β
TRPV1 activation has been shown to exacerbate neuro-inflammatory actions by activating pro-inflammatory STAT3 signaling [29]. Thus, Western blot was used to confirm whether TRPV1 activated JAK2-STAT3 signaling at 24 h after OGD. Western blot results showed that phosphorylation of STAT3 and JAK2 were significantly increased after OGD, which was reversed by either genetic ablation or capsaicin-induced desensitization of TRPV1 (Fig. 3a–c), indicating the critical role of TRPV1 on the phosphorylation of JAK2 and STAT3. Next, we used Stattic, a specific inhibitor of STAT3 phosphorylation, to investigate whether pharmacological inhibition of STAT3 phosphorylation attenuates or abolishes TRPV1-induced increase of GFAP expression. Indeed, treating WT astrocytes with Stattic (5 μM) during OGD challenge significantly reduced the expression of GFAP. In marked contrast, Stattic did not inhibit the expression of GFAP in TRPV1 deficiency astrocytes (Fig. 3d–f).
Recent studies have shown that JAK2-STAT3 signaling was vital for the expression of IL-1β in both resident macrophages [51] and CNS microglia [32], and expression of IL-1β has been used to predict the severity of HIE in infants [12]. Therefore, we next verified the role of TRPV1-activated JAK2-STAT3 signaling in OGD-induced IL-1β upregulation in astrocytes. As shown in Fig. 3g, h, mRNA transcripts and protein level of IL-1β were downregulated by inhibiting the phosphorylation of STAT3 in WT astrocytes and Stattic did not inhibit the expression of IL-1β in TRPV1 deficiency astrocytes. These results suggested that TRPV1 promoted the activation of astrocytes and the expression of IL-1β via JAK2-STAT3 signaling after OGD challenge.
Activation of NLRP3 inflammasome in astrocytes is required for OGD-induced TRPV1-dependent release of IL-1β
The activation of NLRP3 inflammasome was critical for IL-1β release in multiple sclerosis (MS), stroke, and Alzheimer's disease [52]. Following OGD challenge, the levels of mRNA transcripts of NLRP3 inflammasome components (NLRP3, ASC, and caspase-1) increased and the increasing was attenuated by genetic ablation and capsaicin-induced desensitization of TRPV1 (Fig. 4a–c).
ASC is a key adaptor of several inflammasomes including NLRP3 and AIM2, and their activation is reflected by ASC speck assembly or oligomerization [53]. The confocal imaging is used to measure the assembly of ASC speck indicated by the white arrow (Fig. 4d). Upon NLRP3 activation by OGD challenge the dispersed ASC condenses into a large speck in astrocytes, genetic ablation or capsaicin-induced desensitization of TRPV1 reduced ASC oligomerization (Fig. 4d, e). Immunofluorescent staining showed increased intensity (Fig. 4f–h) and co-localization (Fig. 4i) of NRLP3 and caspase-1 after OGD, which were reduced by either genetic ablation of TRPV1 or capsaicin-induced desensitization of TRPV1, suggesting that TRPV1 mediates the release of IL-1β following OGD challenge by activation of NLRP3 inflammasome in astrocytes.
Brain damage and neurobehavioral disorders induced by neonatal HI are reduced in TRPV1 KO mice
IL-1β is the key mediator of neuroinflammation in the CNS [54], and the upper results show that TRPV1 is critical to astrocyte activation and release of astrocyte-derived inflammatory cytokines. To investigate whether the TRPV1-dependent inflammatory response could be translated into brain damage in vivo, we examined brain damage 24 h after neonatal HI in TRPV1 KO mice or WT mice subjected to capsaicin pretreatment. As shown in Fig. 5a, b, the TTC-negative area in the ipsilateral hemisphere was significantly decreased in TRPV1 KO mice and WT mice pretreated with capsaicin (intraperitoneal injection 3 mg/kg 0.5 h before neonatal HI). In marked contrast, post-treating with capsaicin (3 mg/kg, administered 5 min after neonatal HI) did not effectively reduce the TTC-negative area in WT mice (Fig. 5a, b). Next, we used whole-brain Nissl staining to determine the role of TRPV1 in brain atrophy 24 h post neonatal HI (Fig. 5c, d). Neonatal HI produced marked brain atrophy compared with the ipsilateral side of the sham group. Brain atrophy in either TRPV1 KO mice or WT capsaicin pre-treatment mice is significantly reduced. Immunofluorescence showed that the number of IL-1β-positive astrocytes in the ipsilateral hemisphere hippocampus (indicated by the white arrow) decreased in TRPV1 KO mice compared with WT mice after neonatal HI (Fig. 5e, f).
To investigate whether TRPV1 is involved in neonatal HI-induced neurobehavioral disorders, three different neurobehavioral tests including cliff aversion reaction (Fig. 5g), grip test (Fig. 5h) and geotaxis reflex (Fig. 5i) were used. Neonatal HI significantly prolonged the time for WT pups to turn around in the cliff aversion reaction, reduced the holding time in the grip test, and increased the head-turning time in the geotaxis reflex at days 1, 3, and 7 compared with the sham group, which was recovered in TRPV1 KO and WT capsaicin pre-treatment mice. These results suggest that TRPV1 contributes to brain atrophy, infarct size, and loss of reflexes and motor ability caused by neonatal HI.
Both astrocytes and microglia were activated after injury by pro-inflammatory mediators, cytokines, and ROS that are secreted by injured neurons and glial cells [55]. The role of microglia in HIE was well established and microglial activation and aggregation had become one of the hallmarks for HIE in human infants [3, 56,57,58]. Notably, astrocytes are also involved in the pathogenesis of neurological diseases and sterile inflammatory responses [4, 9, 59] and the role of astrocytes in CNS inflammatory was controversial (both detrimental and beneficial) in brain ischemia [3]. Early studies have extensively focused on the protective effects of astrocytes, such as the isolation of the damaged area from the rest of the CNS tissue and reconstruction of the compromised blood-brain barrier [60, 61]. Recent studies, however, demonstrated that astrocytes have a detrimental role in response to ischemic stress [4, 10, 62,63,64].
Ion channels (kv4.1, kv1.3, TRPV1, TRPM7, NKCC1…) and intracellular ion homeostasis (K+, Na+, Ca2+, and Cl−) are critical for glia proper functions in CNS [65,66,67]. TRPV1 that is mainly permeable to Ca2+ naturally expresses in the endomembrane system of microglia and astrocytes which plays important roles in astrocyte and microglial activation [68,69,70]. It has been proved that overactivated TRPV1 increase NADPH oxidase-mediated reactive oxygen species (ROS) generation [71, 72], mitochondrial disruption [73] in microglia, and microglia-induced inflammation [74]. TRPV1 also accelerate astrocyte and microglial migration and chemotaxis during stress by directly interacting with cytoskeletal elements [75, 76]. The role of TRPV1 in microglia is well explored [26], while its distinctive function in astrocyte is not fully understood in a variety of neuroimmune diseases.
In the present study, we showed that during the subacute phase of HI (24 h), astrocyte and microglia were overactivated and microglia activation is about 3 h earlier than astrocyte. This may because microglia acting as the constant sensors of changes in the CNS microenvironment is more sensitive to insults [77]. TRPV1, which is activated after ischemia [78] intensely mediate astrocyte activation compared to microglia, increases IL-1β release and exacerbates ischemia-induced brain damage (brain atrophy, infarct size, and neurobehavioral loss). TRPV1 is required for the phosphorylation of JAK2 and STAT3 which promotes the transcription of GFAP and IL-1β. TRPV1 function is critical for the activation of NLRP3 inflammasome which promotes the cleavage of pro-IL-1β (Fig. 6). Our studies reveal a previously unknown function of astrocytic TRPV1 and potentially identify a critical target for the treatment of CNS inflammation.
Injured cortex becomes hypertrophic in many types of CNS disorders such as stroke and neurotrauma, which is often accompanied with the upregulation of GFAP in astrocytes [9]. As expected, we found that GFAP expression increased in a time-dependent manner after neonatal HI, which is mediated by TRPV1 as either genetic ablation or capsaicin-induced systemic desensitization of TRPV1 inhibited the upregulation of GFAP. In vitro studies further confirmed that TRPV1 directly regulates the function of astrocytes in a cell autonomous manner without the requirement of neurons or microglia, since inhibition of TRPV1 function prevented astrocytes from cellular edema, reduction of branch processes, cell death, and excessive activation caused by OGD challenge.
The role of activated astrocytes has been reported in the sterile inflammatory response and the release of several cytokines, such as IL-1β, IL-6, and TNF [79] in middle cerebral artery occlusion (MCAO). Our previous work demonstrated that TRPV1 activation promotes the expression of TNF, IL-1β, IL-6, and high mobility group box 1 (HMGB1) in repetitive febrile seizure (FS) mice [36]. Consistent with these observations, we found that TRPV1 is required for OGD-induced upregulation of IL-1β, IL-6, and TNF in astrocytes. Interestingly, IL-10 mRNA transcript was slightly increased in a TRPV1-dependent manner after neonatal HI, which might be caused by a negative feedback from the astrocytes induced by severe inflammatory responses [80, 81].
How does TRPV1 mediate astrocyte activation and upregulation of pro-inflammatory cytokines? Previous researches demonstrated that the JAK2-STAT3 pathway is involved in the activation of astrocytes and the expression of cytokines both in vivo and in vitro [30,31,32]. Moreover, in inflammatory signaling cascades in the brain, TRPV1 has been shown to exacerbate neuro-inflammatory actions by activating pro-inflammatory STAT3 signaling [29]. Inhibition STAT3 phosphorylation via JAK2-blockade reduces neonatal HI-induced neuroinflammation and tissue loss [82]. Consistent with these studies, we found that TRPV1 promoted the phosphorylation of JAK2 and STAT3 induced by OGD challenge. Moreover, a specific inhibitor of STAT3 (Stattic) significantly reduced the expression of GFAP and IL-1β following OGD challenge in a TRPV1-dependent manner. These results suggest that TRPV1 mediates the activation of astrocytes and the expression of IL-1β via the JAK2/STAT3 pathway.
IL-1β in umbilical and peripheral blood is noteworthy upregulated in children suffered from HIE, and its expression level corresponds to the grades and adverse outcomes of HIE [83]. The release of IL-1β depends on the activation of NLRP3 inflammasome which cleaves the pro-inflammatory cytokines IL-1β to its active form by mediating the activation of caspase-1 [15, 84, 85]. It has been proved that, in other disease models such as intracerebral hemorrhage model, ischemia/reperfusion injury model, and hemorrhage transgenic amyotrophic lateral sclerosis model, NLRP3 inflammasome is activated in astrocytes [16,17,18,19]. In the present study, the activation of NLRP3 inflammasome followed by IL-1β releasing increased after OGD challenge. And inhibition of TRPV1 blocked this modification, suggesting that TRPV1 drives the release of IL-1β by stimulating the activation of NLRP3 inflammasome after OGD in astrocytes.
In vivo, hampered TRPV1 function significantly reduced brain atrophy, infarct size, and the number of IL-1β-positive astrocytes in the hippocampus. Additionally, the recovery of neurobehavioral disorders indicated by three independent neurobehavioral developmental tests (the geotaxis reflex, cliff aversion reaction, and grip test) after neonatal HI was potentiated in TRPV1 KO mice. Our results suggest that astrocytic TRPV1 is required for the exacerbation of the brain damage and neurobehavioral disorders after neonatal HI. Of note, since TRPV1 do express by microglia/macrophages, neuron [78], and neutrophils [86], these types of cells might contribute to the detrimental effects mediated by TRPV1 independently or cooperatively in HIE. Future studies using cell-specific TRPV1 knockdown/knockout mice needed to confirm this possibility.
TRPV1 promotes activation of astrocytes and release of astrocyte-derived IL-1β mainly via JAK2-STAT3 signaling and activation of the NLRP3 inflammasome. Our findings provide mechanistic insights into TRPV1-mediated brain damage and neurobehavioral disorders caused by neonatal HI and potentially identify astrocytic TRPV1 as a novel therapeutic target for treating HIE in the subacute stages (24 h).
Transient receptor potential vanilloid 1
Glial fibrillary acidic protein
- Iba1:
Ionized calcium-binding adapter molecule 1
Janus kinase 2
Signal transducer and activator of transcription 3
Nucleotide-binding, oligomerization domain (NOD)-like receptor pyrin domain-containing protein 3
Apoptosis-associated speck-like protein containing carboxyl-terminal CARD
Hypoxic-ischemic encephalopathy
Febrile seizure
- IL-1β:
Interleukin 1β
Interleukin 6
Oxygen-glucose deprivation
Phosphate-buffered saline
Tumor necrosis factor
Interleukin 10
2, 3, 5-Triphenyltetrazolium chloride monohydrate
Phenylmethanesulfonyl fluoride
Polyvinylidene fluoride
Bovine serum albumin
Reactive oxygen species
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We thank Meng-Liu Zeng for critically reading and revising the manuscript. We sincerely thank Jun-Chen Liu, Wen-Di Luo, and Jian-Yu Wu for their valuable comments and for the technical assistance during the experiments.
This research is supported by the Natural Science Foundation of China (Grant No. 81571481, No. 81370737, and No. 81601325) and Independent Scientific Research Project Fund of Wuhan University (No. 2042017kf0066).
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YXL, PBW, HXH, and WX conceived and designed the experiments. YXL, WX, MJW, and JGT performed the experiments. YXL, WX, and SL analyzed the data. HXH, LWH, MXY, and HWX contributed to the reagents/materials/analysis tools. YXL, WX, and PBW wrote the paper. All authors reviewed and approved the final manuscript.
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All animals used in experiments were provided by the Nanjing Biomedical Research Institute and the Hubei Province Center for Animal Experiments. And animal experiments were approved by the Care and Use Committee of Wuhan University Medical School.
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The authors declare that they have no competing interests.
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Additional file
Additional file 1:
Knocking out TRPV1-reduced GFAP and Iba-1-positive cell in hypoxia-ischemia brain tissue. (A) GFAP and Iba-1-positive cell were examined in the ipsilateral hemisphere sections from Sham and HI groups. Scale bar = 100 μm. (B, C) The number of GFAP and Iba-1-positive cell in the ipsilateral hemisphere hippocampus. n = 6 for each group. Average values represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Tukey's test after one-way ANOVA and two-way ANOVA). (TIF 8629 kb)
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Yang, XL., Wang, X., Shao, L. et al. TRPV1 mediates astrocyte activation and interleukin-1β release induced by hypoxic ischemia (HI). J Neuroinflammation 16, 114 (2019). https://doi.org/10.1186/s12974-019-1487-3
DOI: https://doi.org/10.1186/s12974-019-1487-3


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UW Science Now is an annual tradition where Town Hall teams with UW Science Engage to bring local graduate students to the stage to present their latest cutting-edge research. We're thrilled to partner with Ada's Technical Books to feature illuminating talks in a casual setting where audiences can enjoy a drink and an evening of scientific breakthroughs!
Our genes make up who we are. They control everything from hair color to height, as well as our susceptibility to certain infections such as Tuberculosis (TB). TB remains a top fatally infectious disease worldwide despite millions of dollars spent on antibiotics to eliminate it. The main problem, says Robyn Emery, is that there is only one vaccine against TB, which is a hundred years old. Emery suggests that a solution to developing a stronger vaccine may lie within our own genes. She presents research on the genes involved in inflammation, exploring the ways they make a person more or less susceptible to TB. By better understanding our bodies' reactions and susceptibility to TB, we can take major steps towards developing a more effective vaccine.
How do our nervous systems tell the difference between a handshake and a hot stove, or an itchy mosquito bite and a painful bee sting? Kali Esancy explores how somatosensory neurons accomplish this task. Esancy shares research on somatosensory neurons—the cells of our nervous systems that detect environmental stimuli like temperature, touch, itch, and pain—highlighting the differences between two groups of neurons that relay itch and pain sensations.
Presented by UW Engage Project, Ada's Technical Books, and Town Hall Seattle.

subset: commoncrawl


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Payroll as a profession has come a long way. A little over a decade ago, as automation swept into the field of human capital management (HCM), some saw the payroll process becoming a cog in the overall machine, absorbed into integrated systems that needed less oversight and skill to manage.
Questions were raised: Was the role of the payroll professional diminishing due to technological advancement? If so, what did that mean for the profession?
It turns out that this payroll automation technology needs more skilled management and oversight than ever. "The dimensions of payroll are very broad and deep," said former American Payroll Association President Linda Obertin, who participated in a recent Source by DailyPay podcast called, "The Future of Payroll."
"The relevancy of the payroll professional is even more critical today," said Obertin, who currently serves as director, Shared Services, at DXC Technology overseeing Global HR and Payroll Services. "Every executive recruiter that I connect with will share their highest-demand requests, and the most difficult roles to fill are in the payroll and control specialist fields."
Technologies that are being developed need to be guided and appropriately adopted into operations. What are some of the key technologies that are certain to be considered in a payroll context?
Dimitris Papageorgiou, a partner at Accenture who assists clients in selecting and deploying global HCM and Payroll solutions, specializes in identifying key technological trends. During the podcast he said on-demand pay, or earned wage access, is "much more mainstream in our days, so many companies are actually now incorporating this as part of their strategy." (DailyPay, sponsor of the Source podcast, is the recognized gold standard on-demand pay provider that uses intelligent automation in its programs.)
Beyond on-demand pay, other significant applications of robotic process automation (RPA), blockchain technology, machine learning or artificial intelligence, natural language processing, radio frequency identification (RFID) and geofencing all are becoming more prevalent in HCM applications. But adoption of these other technologies is mixed, Papageorgiou said.
For RPA and artificial intelligence, applications require operating "within an environment that has quality data and repeatable processes … neither of the two has yet enjoyed broad-based acceptance," Papageorgiou said.
While far behind in adoption, blockchain technology, however, "is going to be the single most impactful disruption in our space," according to Papageorgiou.
"Blockchain in some interesting way is actually contradicting what we're trying to do with AI and RPA," he said. It is a protocol that is "completely going to change the way we think about debits and credits and custody of data."
"My belief is that once blockchain technology matures, a lot of the technology that we utilize today, including in some cases HCM, may not be needed in its present format or present utility," Papageorgiou said.
A payroll professional's part in the transformation of processes will be important.
"As we're thinking of our industry today, we really need to be focused on how technology is evolving our services" to focus on the employee experience, Obertin said. "Employers expect us to have high-quality payroll delivery timeliness, and it's really about how we connect with the partners in the organization to deliver the experience."
"Our role has changed from being more transactional, as Dimitris mentioned, to being more strategic, having a vision, having a plan and executing and being able to measure success, [which are] really, really key skills and strengths for our payroll teams and especially for our payroll leaders."
You can access the full podcast and subscribe to other Source by DailyPay episodes here.
DailyPay, the recognized gold standard for on-demand pay delivery, sometimes called earned-wage access, is a sponsor to the American Payroll Association and also partners with the Bipartisan Policy Center to help bring more awareness of policies that can assist Americans in building financial security.
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A lawsuit window for Pa. survivors of childhood sexual abuse could open as early as next May
State legislative leaders say they'll take a procedural vote on the proposal early next year. 2023 primary voters would then get to weigh in.
This story originally appeared on WITF
State legislative leaders are pledging to vote early next year on a constitutional amendment giving adults who say they survived sexual abuse as children two years to sue their alleged abusers and any institution that sheltered them.
A 2018 Grand Jury report showed the statute of limitations may have closed for hundreds of Pennsylvanians who were sexually abused by Roman Catholic priests as children decades ago. That report recommended lawmakers give all abuse survivors more time to sue.
Pennsylvania now lets anyone who says they were abused as a child come to court with a civil lawsuit before age 55, but some say they missed their opportunity when the time window was smaller.
Research shows social and psychological pressures can keep survivors from coming forward for far longer.
The issue has been a fixture of debate in Harrisburg for years. Bills that would have opened the statute of limitations window fizzled out in previous sessions. Even a state constitutional change, which won approval from both chambers, was derailed when the Department of State bungled its role in the approval process.
Gov. Tom Wolf apologized for the error in a statement Wednesday. The outgoing Democrat threatened to call a special session over the lawsuit window issue in April. Wolf said he's reversing course now that he's secured agreements from the four legislative leaders to "prioritize second passage of a constitutional amendment early next session."
"I am grateful for this agreement so that survivors can seek a path forward toward justice," Wolf said.
In Pennsylvania, the state constitution can be changed only if lawmakers approve an amendment twice in two consecutive sessions. The idea then has to be advertised in local newspapers for a set amount of time before appearing on the ballot during an election. Voters have the final say.
Rep. Jim Gregory (R-Blair County), who is sponsoring the latest lawsuit window proposal, said lawmakers already approved the idea this year – but must now re-approve it before the end of January.
That would give the Department of State enough time to advertise the proposal in newspapers and craft ballot language before the May 2023 primary.
"The most important thing we need to do is get it on the ballot and get people in Pennsylvania to understand why it's important to pass it," Gregory said. "This has everything to do with the victims who are still waiting for their civil justice."
Survivor advocacy groups have voiced frustration with lawmakers for failing to approve a two-year window sooner. Gregory has pinned the blame on former Secretary of State Kathy Boockvar for failing to catch the procedural error that led to the latest delay.
But the Blair County lawmaker acknowledged his own role in an interview Thursday.
"I have to say that I'm sorry because I did not stay on this as closely as I could have," Gregory said. "[But] those are just words, and our actions are what we are most concerned about."
Senate Majority Leader Kim Ward (R-Westmoreland), who has blocked votes on bills that would have opened the lawsuit window, says she supports a constitutional change.
"Without question, all victims of childhood sexual abuse should have the ability to face their abusers," she said in a statement.
Ward added the Senate is "planning to take the next step" in the amendment process once the next legislative session starts in January.
WHYY is your source for fact-based, in-depth journalism and information. As a nonprofit organization, we rely on financial support from readers like you. Please give today.


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Full Gospel Christian Fellowship was founded on August 8, 1978 by the Hull family. Pastor Wilfred Hull pastored the church for many years and to this day has the longest tenure as pastor at Full Gospel. His brother, Raymond Hull served as an interim pastor for 2 years and continues to attend and serve in the church.
Full Gospel Christian Fellowship now pastored by Jesse Knight since July 2019, is a church that preaches Christ. We are a community of believers that love God and love people. We enjoy gathering together to worship, and grow in our faith, but what we enjoy even more is sharing the love of God with those who don't yet know Him as their loving, gracious, merciful and compassionate Father.
When you visit Full Gospel, you can expect to be greeted with love, enjoy a time of Holy Spirit-inspired worship and ministry in the word.
Full Gospel's overseeing credentialing body is Faith Christian Fellowship of Canada.
Our Staff Members
Jesse Knight
Jesse is a husband and a father of three young boys. Jesse is an ordained minister with Faith Christian Fellowship of Canada. He has served in ministry for over 18 years, working as a youth pastor, administrative pastor and itinerant speaker. Pastor Jesse is community & outreach minded. He...


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Aldalifestyle.com – A mesothelioma law firm specializes in asbestos law and mesothelioma cases. Law firms can get compensation for patients and loved ones by filing a mesothelioma lawsuit, asbestos trust fund claim and other benefit claims. National mesothelioma law firms help families nationwide and will often travel to help file a claim.
What Is a Mesothelioma Law Firm?
Mesothelioma law firms focus on asbestos litigation. They specialize in handling personal injury lawsuits, wrongful death lawsuits and trust fund claims for patients and families facing mesothelioma.
The nation's top mesothelioma law firms have years of experience aiding those with suspected asbestos exposure. Teams of specialized legal experts know the best ways to secure compensation to help pay for personalized cancer treatment and other expenses.
Mesothelioma law firms have developed a deep knowledge and emotional understanding of mesothelioma and how stressful it is to navigate cancer treatment and health insurance. They understand how a terminal cancer diagnosis affects families and how important it is to spend precious time with loved ones.
Why You Need a Law Firm Specializing in Asbestos Exposure Cases
Hiring an experienced asbestos law firm is your family's best step toward securing mesothelioma compensation. These specialized firms tend to focus solely on asbestos cases and offer unparalleled expertise in this area of law.
- Streamlined Process: An asbestos firm's background and knowledge translate into a more effortless experience for you and your family throughout the legal process.
- Complex State Laws: Asbestos litigation laws are challenging to understand and vary by state, making a nationwide firm the best way to secure compensation.
- Asbestos Databases: These firms have developed extensive resources to help build your case, including asbestos product databases and historical case records.
- Substantial Rewards: A significant amount of money is at stake in a mesothelioma lawsuit, which can cover treatment costs, end-of-life expenses, travel, lodging and more.
National mesothelioma law firms have litigated cases and trust fund claims nationwide. Choosing a nationwide firm gives you access to a network of attorneys with asbestos case expertise in each state who will determine the proper jurisdiction for filing your claim and maximizing your recovery.
How to Choose a Mesothelioma Law Firm
Mesothelioma law firms specialize in asbestos cases. The first thing to look for when doing your research is their years of experience and a history of success. The top asbestos law firms in the U.S. have a team of qualified attorneys who understand what you and your loved ones are facing and can give you confidence when pursuing your legal options.
The top five must-haves when choosing your mesothelioma law firm are:
Lawyers Who Understand Mesothelioma and Asbestos
Asbestos law firms have intimate knowledge of the different types of asbestos-related diseases and understand how this impacts the patient and the legal process. They know what you and your family are going through and how treatment may affect your ability to attend a trial.
The teams of experts at these firms advocate for expedited trial dates and quick settlements based on the state asbestos laws that apply to your claim. They'll also conduct depositions as soon as possible to avoid statutes of limitations, letting you focus on your medical care.
Ability to Document Asbestos Exposure
Firms specializing in asbestos have a wealth of deep industry resources and proprietary asbestos databases filled with information about asbestos products and contaminated job sites.
Attorneys at asbestos firms will investigate your case in depth to discover proof of your claim. They'll use this information and their extensive databases to connect your mesothelioma diagnosis to prior asbestos exposure.
No Upfront Legal Costs and a Free Case Evaluation
Not every firm offers a free case evaluation, but dedicated mesothelioma law firms typically provide free evaluations, leveraging their nationwide network of attorneys. It is more challenging for local firms to provide this service because they operate on a smaller scale.
The best firms also work on a contingency fee basis, which means you only pay them if they recover compensation for you. You don't pay for their legal services if they don't win your case.
Record of Winning Large Verdicts and Settlements
National firms tend to have the best track record of securing compensation for victims of asbestos exposure. This kind of compensation helps families afford medical treatment, deal with lost wages and handle other expenses such as in-home nursing care.
For example, Weitz & Luxenberg won a multimillion-dollar settlement in 2021 for a Navy veteran who died of mesothelioma. Simmons Hanly Conroy recovered $34 million in 2019 from Ford Motor Company for a mechanic with mesothelioma. Not every case results in multimillion-dollar verdicts, but an experienced firm increases your chance of substantial compensation.
A Lawyer That Makes You Feel Comfortable
Mesothelioma law firms understand that travel is difficult when you've been diagnosed with cancer. One of the most significant benefits of choosing a nationwide firm is its willingness to travel to you or meet virtually, no matter where you live, with more scheduling availability than local firms.
A good legal team will provide examples of its success and explain fees upfront. You'll have time to learn about their attorneys and what makes their firm unique. Mesothelioma attorneys regularly update their clients on progress and often have the experience to anticipate their client's needs.
Working with a Mesothelioma Law Firm
Your attorney will guide you through each step of the lawsuit and help you understand your legal options. They will listen to your concerns and address any questions about the process. The attorney will gather evidence to prepare your case, file a claim in the proper jurisdiction and handle any responses from defendants.
Filing a mesothelioma lawsuit after death is more complicated than a personal injury claim, but a qualified attorney can go over options for filing a wrongful death lawsuit.
What Is the Legal Process Like?
The legal process begins with finding a mesothelioma law firm and scheduling a consultation so you can learn more about the firm and they can learn more about your case.
- Free Consultation: The best mesothelioma law firms offer free consultations and case reviews. During this meeting you can ask questions about the firm's history of success, and the firm will ask you about the patient's diagnosis and work history.
- Documenting Exposure History: If the law firm believes you have a case, they will start to build it by gathering evidence to support your claim. In addition to a lawsuit, they may recommend you file for a trust fund or via the U.S. Department of Veterans Affairs.
- Filing Claims: Once your case is built and supported by evidence, your lawyer will file the claim in the proper state or jurisdiction or with the appropriate asbestos trust fund. Your claim may be valid in multiple states, depending on the statute of limitations.
- Discovery Process: A discovery period will follow for lawsuits, or a claim review will follow for trust fund claims. Your lawyer will handle every aspect of the legal process, and they will coach you through any interviews or depositions you may need to provide.
- Compensation Distribution: If your claim is successful, your lawyer will most likely be the one to distribute compensation to you. You may choose between a lump sum or receiving payments in installments.
If you have questions about the legal process, you should speak with a lawyer to learn more about it and how federal and state laws may impact your case.
Top Mesothelioma Law Firms
In addition to experience and a winning track record, it is important to feel comfortable with the mesothelioma law firm you choose.
Attorneys at top asbestos law firms are accessible, compassionate and sensitive to what you and your family are going through. Your lawyer will manage every aspect of your case, allowing you to focus on what matters most: Treatment and your health.
Weitz & Luxenberg
- Serves Clients: Nationwide
- Years of Experience: 30+
- Free Consultation: In-person and Virtual
- Law Firm Awards: Ranked in the National Tier 1 category of Best Law Firms for mass tort litigation and class actions by U.S. News & World Report.
Since 1986, Weitz & Luxenberg has helped more than 55,000 families affected by asbestos and has won more than $8.5 billion in mesothelioma verdicts and settlements. With offices in New York, New Jersey, Los Angeles and Detroit, the firm handles around 500 asbestos cases yearly with a dedicated team of lawyers, paralegals and support professionals.
Cooney & Conway
- Serves Clients: Nationwide
- Years of Experience: 40+
- Free Consultation: In-person and Virtual
- Law Firm Awards: 2021 Emerging Lawyers Award for Personal Injury Law
Cooney & Conway was founded in 1958 as a personal injury firm in the heart of Chicago. For more than 40 years, the firm has won billions for its clients across thousands of asbestos cases. The firm's multimillion-dollar victories against some of the largest asbestos product manufacturers in the world include United States Gypsum, Armstrong and Owens Corning Fiberglas.
Simmons Hanly Conroy
- Serves Clients: Nationwide
- Years of Experience: 20+
- Free Consultation: In-person and Virtual
- Law Firm Awards: Ranked one of the Best Law Firms in the country by U.S. News & World Report & Best Lawyers
Simmons Hanly Conroy began serving mesothelioma patients and their families in 1999 and has offices in Illinois, New York, California and Missouri. The firm's asbestos team has helped more than 6,000 families and recovered approximately $7 billion in settlements. In 2019, the firm won a $34 million verdict for an 80-year-old mechanic in a four-week trial against Ford Motor Company.
Nemeroff Law
- Serves Clients: Nationwide
- Years of Experience: 25+
- Free Consultation: In-person and Virtual
- Law Firm Awards: 2016 Pound Civil Justice Institute Appellate Advocacy Award
Founder Rick Nemeroff began fighting for individuals in the early 1990s and has offices in Pittsburgh, New Orleans, Utah and Houston. With more than 150 years of collective experience, Nemeroff Law has recovered more than $100 million for clients with victories including a $12.6 million verdict against Owens Corning Fiberglas and $9.25 million against Exxon.
Galiher DeRobertis & Waxman
- Serves Clients: Nationwide
- Years of Experience: 35+
- Free Consultation: In-person and Virtual
- Law Firm Awards: Leading mesothelioma law firm for the Hawaiian Islands
Galiher DeRobertis & Waxman is based in Honolulu but has traveled to more than 40 states to meet with clients. The team has unique litigation resources, including an extensive database of job sites, asbestos products and an archive of Navy records. After more than 250 jury verdicts, the firm has recovered more than $1 billion for victims.
State Legal Directories: Mesothelioma Lawyers Near Me
Asbestos litigation laws vary by state, and many of the nation's top mesothelioma law firms are licensed to practice in multiple states throughout the U.S. Knowledge of local asbestos laws and state statutes of limitations are essential to winning cases in any jurisdiction.
- California
- Florida
- Georgia
- Illinois
- New York
- Ohio
- Pennsylvania
- Texas
Firms that practice nationwide have handled cases in many states, giving them experience processing claims both nationally and on a state level. This expertise helps them develop substantial lawsuits that move smoothly through the legal system.
Common Questions About Hiring a Mesothelioma Law Firm
What's the difference between a law firm specializing in asbestos and one that doesn't?
An asbestos law firm specializes in asbestos law and mesothelioma cases. These firms typically employ mesothelioma lawyers with years of experience in asbestos cases and a track record of successful outcomes. A law firm that doesn't specialize in asbestos won't have access to the best asbestos databases and may incorrectly file complicated claims, which could delay or significantly reduce the compensation received.
Can a family member hire a law firm for an asbestos case?
Yes, legal decisions after the death of a mesothelioma patient fall to the estate representative. The estate representative can be a family member or friend named by the decedent or appointed by the court. Family members can file an asbestos claim after death in a wrongful death lawsuit. Rules and limitations for wrongful death claims vary by state.
How long do I have to hire a mesothelioma law firm?
The time limits on mesothelioma cases vary by state law and are known as statutes of limitations. These statutes often range from 12 to 36 months after the date of a mesothelioma diagnosis for personal injury claims. The period for wrongful death claims usually begins when an asbestos disease is ruled as a patient's cause of death. Please make sure you speak with an attorney specializing in your type of claim to get specific answers about the applicable statute of limitations.


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The UN High Commissioner for Human Rights called on Friday for an independent war crimes investigation in Gaza after reports that Israeli forces shelled a house full of Palestinian civilians, killing 30 people.
Navi Pillay told an emergency meeting of the UN Human Rights Council that the harm to Israeli civilians caused by Hamas rockets was unacceptable, but did not excuse any abuses carried out by Israeli forces in response.
Pillay went further in an interview with the British Broadcasting Corp., saying an incident in Gaza City this week "appears to have all the elements of war crimes."
The UN Office for the Coordination of Humanitarian Affairs said Israeli troops evacuated Palestinian civilians to a house in the Zeitoun neighborhood on Jan. 4, then shelled the building 24 hours later.
The UN agency said 110 people were in the house, according to testimony from four witnesses.
On Thursday, the international Red Cross said the Israeli army refused rescuers permission to reach wounded people in the neighborhood for four days. Israel said the delay was caused by fighting in the area.
Pillay told the Geneva-based rights council that all parties to the conflict had a duty to care for the wounded and avoid targeting health workers, hospitals and ambulances.
Violations of international humanitarian law may amount to war crimes for which individuals should be held accountable, she said.
The 47-member council, which is dominated by Arab and African countries, is debating a resolution condemning Israel for its actions in Gaza. The motion could be delayed until Monday.


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Military Makeover With Montel issued the following:
27 Year Marine Veteran Receives Complete Home Renovation Thanks to National TV Show Military Makeover with Montel, WWE Superstar Lacey Evans and Brand Partners.
Tune in August 21st for season 23 premiere on Lifetime at 7:30am EST – The Military Makeover team renovated the home of Marine Corps Veteran Marcelino Marquez in Dacula, GA. All aired episodes can be viewed at www.militarymakeover.tv.
DEERFIELD BEACH, FL – In true Military Makeover style, host Montel Williams, cast and crew, special guest WWE Superstar Lacey Evans, and equally passionate national brands have come together again to serve those who have served. Marine Corps Veteran Marcelino Marquez along with his family will be the recipients of a beautifully renovated home among other items of gratitude. The first of five (5) episodes airs on August 21st at 7:30am EST. All aired episodes can be viewed at militarymakeover.tv.
Marcelino Marquez enlisted out of high school and rose to one of the highest enlisted ranks of Master Gunnery Sergeant. Marcelino deployed on two tours in Iraq, two tours in Afghanistan, along with many other challenging postings. He started as an infantryman, then became a mortar man before shifting from mortars to close air support as a Low Altitude Air Defense (LAAD) Gunner. Marcelino later transitioned into Mortuary Affairs where he was tasked with the search, recovery, identification, preparation and return to the US of American and American-allied personnel killed in action. Those final emotionally wrenching assignments have left him suffering from post-traumatic stress disorder (PTSD). Marcelino with his wife Francis, and three children, have been selected as our new Military Makeover family.
The Military Makeover team partners up with likeminded brands with a similar mission to help veterans such as our ongoing sponsors: Goodman Manufacturing, ABC Supply Co, MyComputerCareer, Tarkett and CaptionCall. This process also involves local businesses in the community and invites volunteers to step up and lend a hand in support of the Marquez family throughout the renovation process. Special guest WWE Superstar Lacey Evans, a veteran of the U.S. Marines, makes an appearance and supports the Marquez family throughout the renovation process. The final reveal unites cast, brand partners and volunteers, creating a literal "community celebration" of support for the change they created together.
"In the nearly three decades since I retired from the Navy, I've never really taken the uniform off because standing up for those who are serving now and those who have served has been the greatest honor of my professional career." – Montel Williams, Host and Co-Executive Producer
"We are so fortunate to be able to cultivate strong partnerships with national brands, non-profits, and local military communities to make each makeover better than the next."- Mark Alfieri, Founder and CEO of BrandStar
About Military Makeover With Montel
Military Makeover with Montel®, A BrandStar Original, is America's Leading Branded Reality TV Show that offers hope and a helping hand here on the home front to members of our military and their loved ones. A veteran of both the Marine Corps and the Navy, talk show legend and military advocate Montel Williams, who creatively co-produces the show along with a colorful cast that seeks to transform the homes and lives of military families across the country. The cast includes co-host Art Edmonds, designer Jennifer Bertrand and contractor Ryan Stanley. This special series enlists caring companies of all sizes as well as non-profits and the local community. Military Makeover airs on Lifetime® and on the American Forces Network which serves American servicemen and women, Department of Defense and other U.S. government civilians and their families stationed at bases overseas, as well as U.S. Navy ships at sea. Help starts at home for veterans on Military Makeover. Join us as our makeover team engages to change the living situation – and the lives – of these deserving families.


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The first topic in CX Club is CX Principles and in this video we look at the first three of eleven principles:
A. CX isn't something you do… – CX, unlike the other disciplines of the business, is not a function. It's purely about what the customer 'gets' and is an outside-in view rather than inside-out. It's about understanding the experience of the customer in terms of their interaction with the product, service and channel (sales, marketing & distribution) propositions of the organisation; and then finding ways of improving the experience to better meet the wants and needs of the customer.
B. CX isn't Customer Service "on acid"! – A representative from the Institute of Customer Experience once described CX as "Customer Service on acid" and was taken to task by a group of CX professionals, who explained that the experience of the customer comes from interactions with the product, channel AND service propositions of the business, so is much larger than Customer Service
C. CX, journeys and stories – CX is unique, as it takes a chronological look at the relationship between the customer and the organisation. Organisations don't often the look at themselves chronologically, but customers do all the time. When judging their experiences, they consider all interactions with the organisation over time, hence the importance of Customer Journeys. Stories are important too, as they highlight the emotional nature of experience. Chronology drives recollection, recollection drives nostalgia, nostalgia drives passion and passion drives relationships.
In the next video, we continue looking at CX Principles with:
D. CX is only as strong as the weakest point in the customer journey
E. Consistency is key
F. The role of the brand
G. CX is the gap between what the customer gets and what they expected to get
See you next time…


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$ Refer to Invoice
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With your trip date approaching, please use the form below to submit your payment information.
Please let me know if you have any questions.
If you do NOT know your reservation number, please notate the destination you are paying for. If you are paying for someone else, please notatate their name in the reservation number section. Please allow up to 2 business days for payment to be processed.


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On Feb. 12, President Trump released his 55-page outline for legislation to establish several new infrastructure funding programs, streamline the regulatory review process for projects, improve workforce development programs, and set other federal infrastructure priorities.
The "Legislative Outline for Rebuilding Infrastructure in America" describes
- a new, $50 billion program to promote investments in various forms of infrastructure including drinking water, wastewater, and stormwater;
- a new, $20 billion financial assistance program administered by the U.S. Department of Commerce for infrastructure-related projects;
- $20 billion for expanding several existing federal infrastructure-financing programs such as the Water Infrastructure Finance and Innovation Act (WIFIA);
- proposals to open the Clean Water State Revolving Fund to allow private entities to receive loans through the program;
- reductions in the level of federal regulatory review for a project when a state or private entity provides the majority of project funding; and
- amendments to the Clean Water Act to ensure privately owned treatment works have regulatory requirements identical to those for publicly owned treatment works.
The Water Environment Federation (WEF; Alexandria, Va.) would like potential infrastructure initiatives to
- expand meaningful assistance to states and local governments;
- build resilient water infrastructure;
- facilitate innovation in the water sector; and
- promote workforce development through training, internships, apprenticeships, and career-pathway programs.
"We are at a pivotal time, with aging and inadequate water infrastructure in desperate need of repair and expansion to meet the challenges and seize the opportunities for water in the 21st century," said Eileen O'Neill, executive director of WEF. "We look forward to working with the administration and Congress on a legislative package that addresses our nation's pressing and long-term water infrastructure needs."
Members of the U.S. House of Representatives and U.S. Senate also are developing their own infrastructure plans. WEF encourages its members to write to Congressional representatives to urge them to include funding for clean water, drinking water, and stormwater infrastructure in any related future legislation.
Closing the gap between needed and appropriated water infrastructure investments would generate more than $220 billion in annual economic activity and generate 1.3 million jobs over 10 years, according to the report "The Economic Benefits of Investing in Water Infrastructure."
Infrastructure funding will be a key topic for a discussion during the 2018 National Water Policy Fly-In, April 17–18 in Washington, D.C., during Water Week 2018. Attending WEF members will have an opportunity to talk to their members of Congress about these topics.


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The problem
In 2016, we improved the acoustics in the HR-office of Konvert Services in Courtrai with a lot of success. A year later, in 2017, we were contacted by this satisfied client to improve the acoustics in the office located in Tournai. The large windows gave the impression that the consultation room would be pleasant to work in, due to it being nice and bright in there. However, this impression didn't take the irritating reverberations into account that were enhanced by multiple expanses of glass.


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2008 is well under way, and time to continue to our look at this year market statistics for the area. As we have always stated that as a homeowner, potential resident, potential seller, or as a real estate consultant, we think it is always important to know the stats about what is happening with Mountain Brook AL Real Estate
March 2008 Listed Properties in Mountain Brook Alabama
For the month of March there were 61 Newly Listed Properties. The Average Price was $807,187. The Median Price was $549,900, and the Price Range was $240,000-$2,950,000.
March 2008 Sold Properties in Mountain Brook Alabama
For the month of March there were 19 Sold Properties. The Average Price was $679,374. The Median Price was $506,000, and the Price Range $245,000-$1,900,000 with 55 Average Days on Market.
Currently active as of 4/8/08 in Mountain Brook Alabama
Currently there are 197 Active Properties with an Average Price of $914,504. The Median Price is $599,774 and the Price Range is $240,000-$5,800,000 with 96 Average Days on Market.
If you would like to be contacted as soon as homes become available in the Mountain Brook area just let me know, or if you would like to see statistics about other areas, don't hesitate to ask.
To see how much your Mountain Brook Home may be worth visit MountainBrookHomePrices.com
To search all of the homes for sale in Mountain Brook and all its communities visit MountainBrookHomeSearch.com
*Note these stats are accurate as of 4/8/08 according to the Birmingham MLS. They are subject to change as agents submit listed and sold properties into the MLS


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I have not run away from being your shepherd, nor have I desired the day of sickness. You know what came out of my lips; it was before your face.
for I did not shrink from declaring to you the whole counsel of God.
how I did not shrink from declaring to you anything that was profitable, and teaching you in public and from house to house,
My anguish, my anguish! I writhe in pain! Oh the walls of my heart! My heart is beating wildly; I cannot keep silent, for I hear the sound of the trumpet, the alarm of war. Crash follows hard on crash; the whole land is laid waste. Suddenly my tents are laid waste, my curtains in a moment.
Not many of you should become teachers, my brothers, for you know that we who teach will be judged with greater strictness.
Know this, my beloved brothers: let every person be quick to hear, slow to speak, slow to anger;
For we are not, like so many, peddlers of God's word, but as men of sincerity, as commissioned by God, in the sight of God we speak in Christ.
For our boast is this, the testimony of our conscience, that we behaved in the world with simplicity and godly sincerity, not by earthly wisdom but by the grace of God, and supremely so toward you.
I am speaking the truth in Christ—I am not lying; my conscience bears me witness in the Holy Spirit— that I have great sorrow and unceasing anguish in my heart. For I could wish that I myself were accursed and cut off from Christ for the sake of my brothers, my kinsmen according to the flesh.
Then Amos answered and said to Amaziah, "I was no prophet, nor a prophet's son, but I was a herdsman and a dresser of sycamore figs. But the Lord took me from following the flock, and the Lord said to me, 'Go, prophesy to my people Israel.'
"So you, son of man, I have made a watchman for the house of Israel. Whenever you hear a word from my mouth, you shall give them warning from me. If I say to the wicked, O wicked one, you shall surely die, and you do not speak to warn the wicked to turn from his way, that wicked person shall die in his iniquity, but his blood I will require at your hand. But if you warn the wicked to turn from his way, and he does not turn from his way, that person shall die in his iniquity, but you will have delivered your soul.
The Spirit lifted me up and took me away, and I went in bitterness in the heat of my spirit, the hand of the Lord being strong upon me. And I came to the exiles at Tel-abib, who were dwelling by the Chebar canal, and I sat where they were dwelling. And I sat there overwhelmed among them seven days. And at the end of seven days, the word of the Lord came to me: "Son of man, I have made you a watchman for the house of Israel. Whenever you hear a word from my mouth, you shall give them warning from me. If I say to the wicked, 'You shall surely die,' and you give him no warning, nor speak to warn the wicked from his wicked way, in order to save his life, that wicked person shall die for his iniquity, but his blood I will require at your hand.
If I say, "I will not mention him, or speak any more in his name," there is in my heart as it were a burning fire shut up in my bones, and I am weary with holding it in, and I cannot.
Should good be repaid with evil? Yet they have dug a pit for my life. Remember how I stood before you to speak good for them, to turn away your wrath from them.
"You shall say to them this word: 'Let my eyes run down with tears night and day, and let them not cease, for the virgin daughter of my people is shattered with a great wound, with a very grievous blow. If I go out into the field, behold, those pierced by the sword! And if I enter the city, behold, the diseases of famine! For both prophet and priest ply their trade through the land and have no knowledge.'" Have you utterly rejected Judah? Does your soul loathe Zion? Why have you struck us down so that there is no healing for us? We looked for peace, but no good came; for a time of healing, but behold, terror. We acknowledge our wickedness, O Lord, and the iniquity of our fathers, for we have sinned against you. Do not spurn us, for your name's sake; do not dishonor your glorious throne; remember and do not break your covenant with us.
But if you will not listen, my soul will weep in secret for your pride; my eyes will weep bitterly and run down with tears, because the Lord's flock has been taken captive.
Oh that my head were waters, and my eyes a fountain of tears, that I might weep day and night for the slain of the daughter of my people!
Now the word of the Lord came to me, saying, "Before I formed you in the womb I knew you, and before you were born I consecrated you; I appointed you a prophet to the nations." Then I said, "Ah, Lord God! Behold, I do not know how to speak, for I am only a youth." But the Lord said to me, "Do not say, 'I am only a youth'; for to all to whom I send you, you shall go, and whatever I command you, you shall speak. Do not be afraid of them, for I am with you to deliver you, declares the Lord."
Unless otherwise indicated, all content is licensed under a Creative Commons Attribution License. All Scripture quotations, unless otherwise indicated, are taken from The Holy Bible, English Standard Version. Copyright ©2001 by Crossway Bibles, a publishing ministry of Good News Publishers. Contact me: openbibleinfo (at) gmail.com.


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Education Minor
Please Note:
Programs in the Department of Education are not admitting new students.
Students currently enrolled in the program will be allowed to complete
the program requirements as defined by each student's plan of work.
Minor in Education
The 18-credit-hour minor in Education is designed for those students who are interested in applying content knowledge in diverse settings and working with diverse populations. The coursework is based on the Education Department's Conceptual Framework which addresses the ethical behaviors for demonstrating how a teacher as scholar, inquirer and moral agent interacts with their constituents in the role of educator.
Education Minor Required Courses
Program Contact Information
Department Chair: Alan Grigg, Ed.D.
Reno Hall, Room 243
McNichols Campus
Email: firstname.lastname@example.com
Telephone: 313-993-1475
Fax: 313-578-0507