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Combination of isoflurane and propofol, a means for general anesthesia in the surgery of perioperative cerebral hypoperfusion rats to avoid cognitive impairment CURRENT STATUS:

Background: Perioperative cerebral hypoperfusion often occurs. However, the underlying mechanism and targeted interventions remain mostly to be determined. Anesthetic isoflurane induces neuronal injury via endoplasmic reticulum (ER) stress, whereas sub-anesthetic dose of propofol improves postoperative cognitive function. However, the effects of the combination of isoflurane plus propofol, which is a common combination of anesthesia for patient, on ER stress and the associated cognitive function remain unknown. Methods: We therefore set out to determine the effects of isoflurane plus propofol on the ER stress and cognitive function in the rats insulted by cerebral hypoperfusion. The rats received isoflurane alone (1.9%), propofol alone (40 mg·kg -1 ·h -1 ) or a combination of isoflurane and propofol (1% and 20 mg·kg -1 ·h -1 or 1.4% and 10 mg·kg -1 ·h -1 ). Behavior studies (Fear Conditioning test) and biochemical analyses (Nissl staining and western blotting for the harvested rat brain tissues) were employed in the studies. Results: We found that the combination of 1% isoflurane plus 20 mg·kg -1 ·h -1 propofol attenuated the cerebral hypoperfusion-induced cognitive impairment and the ER stress. Conclusions: These data suggest that ER stress contributes to the underlying mechanism of cognitive impairment and the combination of isoflurane and propofol was able to preserve cognitive function in the rats after cerebral hypoperfusion via prevention of ER stress. These findings have established a system to study the strategy in preventing and treating perioperative cerebral hypoperfusion, leading to promotion of the future larger scale studies.

To observe the effects of different dosages of isoflurane and propofol on cognitive function, a contextual fear conditioning test was performed on the first and seventh days after ORIF. The percentage of freezing time in Group C and Group IP 1 was not significantly different on both the first day (C vs IP 1 : 44.23 ± 6.60 vs 42.86 ± 7.12, P = 1.00) and seventh day (C vs IP 1 : 35.70 ± 5.21 vs 34.85 ± 5.02, P = 1.000) after ORIF ( Fig. 2A). However, in Group IP 2 , I and P, the percentage of freezing time was much lower than that of Group C on Day 1 (all P < 0.05) and Day 7 (all P < 0.05) ( Fig. 2A). The results suggest that combination of 1% isoflurane and 20 mg·kg -1 ·h -1 propofol could protect cognitive function while other dosages couldn't.
Treatment with isoflurane or propofol alone induced irreversible neuronal injury in the hippocampus

CA1 region
Hippocampal slices were stained with cresyl violet (Nissl-staining method) to investigate potential neuronal damage caused by anesthetics on days 1 and 7 after ORIF. Compared with group C, the number of surviving neurons decreased one day after ORIF only in Group I (C vs I: 193.13 ± 23.94 vs 150.88 ± 20.19, P = 0.039, Fig. 2 B-C). On the seventh day after ORIF compared with Group C, the number of surviving neurons was significantly down-regulated in groups I and P (C vs I: 187.38 ± 19.86 vs 146.75 ± 16.70, P = 0.008; C vs P: 187.38 ± 19.86 vs 173.35 ± 28.99, p = 0.011).
No significant change was found in Group IP 1 and IP 2 . (Fig. 2

B-C)
Combination treatment with 1% isoflurane and 20 mg·kg -1 ·h -1 propofol maintained the expression level of cell GABA A R α1 in the hippocampus As mentioned above, GABA A R α1 is a key subunit that affects cognitive function. Therefore, western blotting was performed on the first and seventh days after ORIF to evaluate the expression of GABA A R α1 subunit. There was no difference in the expression of GABA A R α1 between group C and group IP 1 on day 1 (C vs IP 1 : 100.00 ± 18.48 vs 91.86 ± 15.45, P = 0.629) or day 7 (C vs IP 1 : 100.00 ± 14.72 vs 112.39 ± 20.17, P = 0.261) after ORIF. The expression of GABA A R α1 were down-regulated on days 1 and 7 after ORIF in Group IP 2 , I and P compared with group C (all P < 0.005, Fig. 3).
Combination treatment with 1% isoflurane and 20 mg·kg -1 ·h -1 propofol protected neurons from apoptosis To analyze ER related apoptosis, expression of CHOP was valued by western blotting. There was no difference between group C and group IP 1 on day 1 (C vs IP 1 : 100.00 ± 13.63 vs 76.93 ± 13.74, P = 0.409) and day 7 (C vs IP 1 : 100.00 ± 20.70 vs 82.77 ± 11.96, P = 0.876). Compared with Group C, the expression of CHOP in group IP 2 didn't change obviously on the first day (C vs IP 2 : 100.00 ± 13.63 vs 136.70 ± 17.07, P = 0.058) but increased markedly on the seventh day after ORIF (C vs IP 2 : 100.00 ± 20.70 vs 191.85 ± 37.16, P < 0.001). The expression of CHOP was significantly up-regulated on both day 1 and day 7 after ORIF in the I and P groups (all P < 0.005). (Fig. 4) 1% isoflurane and 20 mg·kg -1 ·h -1 protects neurons by heightening expression of BiP The expression of BiP in Group IP 1 , IP 2 , I and P was all up-regulated compared with Group C on Day 1 (all P < 0.005). The highest was Group IP 1 (C vs IP 1 : 100.00 ± 18.58 vs 442.86 ± 69.09, P < 0.001) and the lowest was Group I (C vs I: 100.00 ± 18.58 vs 165.13 ± 25.53, P < 0.001). On Day 7, the expression of BiP fell down in all four groups and there was no difference between Group I and Group C (C vs IP 1 : 100.00 ± 13.91 vs 142.57 ±27.93, P = 0.053). However, in Group IP 1 , IP 2 and P, it was still much higher than Group C (all P < 0.05, Fig. 5). The highest one was still Group IP 1 (C vs IP 1 : 100.00 ± 13.91 vs 268.27 ± 46.51, P < 0.001). (Fig. 5)

Discussion
CH has been universally acknowledged as a risk factor of perioperative stroke, whose incidence ranges of 0.1%-7.9%[17]. Significant CAS has an independent impact on CH and cognitive deficits to various degrees [4]. Given that patients with perioperative CH (especially caused by CAS) are often observed, it is necessary to evaluate the effects of general anaesthesia on this kind of patients. To mimic the pathological process of CAS, we ligated the bilateral CCA of rats 30 days before the ORIF surgery. After the ORIF surgery, behaviour experiments (FC test) and biochemical analyses (Nissl staining and western blotting for the harvested rat brain tissues) were carried out to evaluate cognitive function of rats.
FC test is a very sensitive and effort-independent test of learning and memory [18]. In order to eliminate effects on motor ability caused by tibial fracture, FC test was chosen to inspect cognitive function after surgery of ORIF. Isoflurane has been reported to suppress learning in a dose-dependent fashion. Hence, we trained animals before surgery and anesthesia to remove the influence of the acquisition phase on assessment of memory postoperatively [19]. Our results suggest that memory wasn't impaired only in rats anesthetized with 1% isoflurane and 20 mg·kg -1 ·h -1 propofol. Such obvious difference aroused our interests in detecting the state of related anatomic structures.
Hippocampal CA1 area is crucial for context-specific memory retrieval and spatial memory. After CA1 lesions, both recent and remote memory is impaired [20]. More than that, this area is vulnerable to ischemia injury [21]. Thus, we chose hippocampal CA1 area to measure the number of survival neurons and expression of certain protein. Result of survival neurons wasn't coincident with our anticipation. We speculate that although neurons are still alive, some cognitive-related functional structures may have changed, which in turn affects behavioral outcomes. Or perhaps some of the damage has already occurred during anesthesia, but it needs time to accumulate enough to kill the neurons. This may explain why the number of neurons in Group P was reduced on the Day 7 not Day 1. Of course, we can't ignore the subjective judgment error caused by the experimental method, which needs manually counting the number of Nissl body. Nevertheless, we can still draw a conclusion from the results that, compared to combination groups, the high dose of isoflurane or propofol alone can cause irreversible damage to the nervous system. GABA A R α1 subunit has also been linked to brain cognitive functions [12]. More recently the expression level of GABA A R α1 in the hippocampal CA1 region was found significantly down-regulated in rats with chronic ischemic encephalopathy [22]. In our study, expression of GABA A R contains α1 subunit decreased in all but one group (1% isoflurane and 20 mg·kg -1 ·h -1 propofol). It indicates that improper usage of anesthetics could aggravate cognitive impairment even though neurons are alive.
Previous study has confirmed that isoflurane affects cognitive function by ER stress [6]. Expression of BiP and CHOP are evidence of heightened ER stress [23,24]. Prolonged or excess CHOP expression has been accepted as key to ER stress-related apoptosis [16]. In our study, 1% isoflurane and 20 mg·kg -1 ·h -1 propofol didn't cause CHOP activation on neither one nor seven days after administration of anesthetics, while other ways of general anesthesia increased expression of CHOP variously. BiP normally alleviates the UPR and is anti-apoptotic [25]. In our experiments, the expression of BiP in rats anesthetized with 1% isoflurane and 20 mg·kg -1 ·h -1 propofol was the highest among all these four general anesthesia groups. The results indicate that highly increased should be the key for 1% isoflurane and 20 mg·kg -1 ·h -1 propofol to protect cognitive function from deterioration.
Based on previous studies and our experiments, we can get the following inference. Anesthetics stimulate neurons and cause accumulation of unfolded or misfolded proteins in ER, thereby induce ER stress. BiP dissociates with transducers of unfolded protein response (UPR) to transport unfolded/misfolded proteins to cytoplasm and trigger ER associated degradation (ERAD) [26].
Meanwhile, the transcription and translation of proteins within neurons except for UPR downstream biomarkers are inhibited. The expression of BiP and CHOP increases due to UPR. If the increased expression of BiP is sufficient to bind unfolded/misfolded proteins, then ER stress could be alleviated and the neuron could adapt. If not, prolonged or excess expression of CHOP will lead to apoptosis [27]. Even though in some cases the stimulation is not severe enough to cause apoptosis, it still can cause cell defects when the transcription and translation of some functional proteins, for example GABA A R α 1 subunit, are suppressed. (Fig.6) The advantage of this experiment is that we selected rats with CH as objects. The cognitive function of CH rats is more vulnerable and more sensitive to injury stimuli than that of normal rats. If we choose normal rats as research objects, some subtle difference may be ignored because of their strong adaptive ability. Combination of anesthetics selected by our experiments is especially important for patients with CH but it is also necessary for patients with normal cognitive function.
Our experiments also bring new inspiration to basic researchers. To dates, trying to find the critical point between UPR-induced cell adaptation and apoptosis has been a center of attention. In our study, we have showed both two outcomes. Our grouping model can provide a reference for researchers when they want to select stimulus factors.
The downside of our research is that only the effects of the anesthesia maintenance process have been studied. For the same drug compatibility, if the way of induction of anesthesia changed, will the results be different? For the pathways mentioned above, the specific changes of the midstream molecules remain to be discovered.

Conclusion
In conclusion, we can draw a conclusion that 1% isoflurane and 20 mg·kg -1 ·h -1 propofol is the best way to avoid further damage to cognitive function of CH rats. The underlying mechanism of this phenomenon is related to alleviation of ER stress.

Methods
In our study, a ligation of bilateral CCA (common carotid arteries) surgery [28] was adopted to prepare rats as CH animal model [29,30]. A second surgery, open reduction and internal fixation (ORIF) [31], requiring general anesthesia, was operated so that the effects of anesthetics on cognitive function of these CH rats could be assessed (Fig. 1).

Ligation of CCA
The rats were first anaesthetized with intraperitoneal (i.p.) injection of 10% thiobutabarbital (100 ml/kg). After the body motion reaction and righting reflex disappeared, rat was fixed on the operation platform. Throughout the whole procedure, the surgical field was maintained sterile. The skin of the rat's neck was shaved and disinfected with iodine tincture. A median incision of approximately 2-3 cm was made in the neck. The muscles and surrounding tissues were separated to expose CCA. The CCA and a blunt end syringe needle (0.45 mm in diameter, 1 cm in length) were ligated tightly at the proximal side 1.5 cm from the bifurcation of the internal and external carotid arteries. The slipknot was firmly fixed and the needle was carefully removed. The wound was sutured and disinfected.
During the surgery, a heating lamp was used to help maintaining the body temperature of anesthetized rats at 37 ± 0.5°C. [28] Anesthesia and surgery of ORIF During the ORIF surgery, rats were administered isoflurane inhalation or propofol through the tail vein. For the induction phase of anesthesia, rats were placed in a transparent chamber (W 25 cm × D 15 cm × H 10 cm) connected to a vaporizer and anaesthetized with 5% isoflurane and 40% oxygen.
When the rats' righting reflex disappeared, the chamber was replaced by a mask. Then all the rats were separated into 5 groups (n=32/group) and given different anesthesia maintenance methods.   Combination treatment with 1% isoflurane and 20mg·kg-1·h-1 propofol maintain the expression of GABAAR α1 subunit. (A-C) The expression of GABAAR α1 subunit in hippocampus was determined by western blotting on Day 1 and Day 7 after ORIF. Data was expressed as mean ± SD (n=8/group). * P < 0.05 compared with group C; # P < 0.05 compared with group IP1; ^ P < 0.05 compared with group IP2. Combination treatment with 1% isoflurane and 20mg·kg-1·h-1 propofol avoided ER related apoptosis. (A-C) The expression of CHOP in hippocampus was determined by western blotting on Day 1 and Day 7 after ORIF. Data was expressed as mean ± SD (n=8/group). * P < 0.05 compared with group C; # P < 0.05 compared with group IP1; ^ P < 0.05 compared with group IP2 Figure 5 Combination treatment with 1% isoflurane and 20mg·kg-1·h-1 propofol maintain neurons adaptive ability by increasing expression of BiP. (A-C) The expression of BiP in hippocampus was determined by western blotting on Day 1 and Day 7 after ORIF. Data was expressed as mean ± SD (n=8/group). * P < 0.05 compared with group C; # P < 0.05 compared with group IP1; ^ P < 0.05 compared with group IP2 Figure 6 Combination treatment with 1% isoflurane and 20mg·kg-1·h-1 propofol protects neurons from ER stress. (1) 1% isoflurane and 20mg·kg-1·h-1 propofol promotes more expression of BiP than other groups. (2) 1% isoflurane and 20mg·kg-1·h-1 propofol didn't induce expression of CHOP while other groups did.

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