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

Background: Perioperative cerebral hypoperfusion often occurs. However, the underlying mechanism of cognitive impairment resulting when perioperative cerebral hypoperfusion occurs 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. A ligation of bilateral common carotid arteries (CCA) surgery was adopted to prepare rats as cerebral hypoperfusion (CH) animal model. A second surgery, open reduction and internal fixation (ORIF), requiring general anesthesia, was operated 30 days later so that the effects of anesthetics on cognitive function of these CH rats could be assessed. 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), histological analyses (Nissl staining) and biochemical analyses (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 propol did not aggravate the cognitive impairment and the ER stress in aging rats with cerebral hypoperusion and being subjected to an ORIF surgery. Conclusions: These data suggest that ER stress contributes to the underlying mechanism of cognitive impairment and the combination of isoflurane and propofol did not aggravate the cognitive impairment and the ER stress in aging rats with cerebral hypoperfusion and being subjected to an ORIF surgery.

has been well known related with neurodegenerative diseases [24,25]. The unfolded protein response (UPR) triggered by ER stress is an important quality control system for maintaining protein homeostasis (Proteostasis). Proteostasis refers to an equilibrium state of specific protein synthesis, folding and unfolding, modification and degradation in the intracellular proteome at a specific time point. The ER of the cell is a site for the folding and post-translational processing of secreted proteins and membrane proteins (about 1/3 of the human proteome). Binding immunoglobulin protein (BiP), also known as glucoseregulated protein 78 (GRP78), is an ER chaperone protein whose expression is part of the UPR, is required to alleviate ER stress [26]. Once ER stress occurs, Bip binds to unfolded proteins and activates the downstream receptor proteins, increasing molecular chaperone expression, reducing global protein translation, and increasing unfolded/misfolded proteins. It degrades and reduces ER stress and protects cells through endoplasmic reticulum-associated degradation (ERAD).
The expression of C/EBP homologous protein (CHOP), a transcriptional factor, is also induced by ER stress but indirectly regulates apoptosis [27]. During a stress situation, UPR attempts to increase protein-folding capacity and remove misfolded and unfolded proteins. If the remedy is inadequate to restore homeostasis under chronic ER stress, terminal UPR will trigger apoptosis through abundant signaling mechanisms, mainly mediated by CHOP, c-Jun N-terminal kinase (JNK), and caspase-12, with CHOP as the most widely studied [28].
Thus, the expression of BiP, CHOP and GABA A R α1 subunit was used to evaluate cellular mechanisms accounting for neural substrate conditions allowing normal cognitive functions in this study.
The objective of the current studies is to explore ways of general anesthesia for rats with CH and being subjected to an ORIF surgery to protect cognitive function. We tested a hypothesis that combination of isoflurane and propofol is better to protect cognitive function than single usage of isoflurane or propofol during ORIF surgery by using behavior studies and biochemical analyses.

Results
Combination treatment with 1% isoflurane and 20 mg·kg -1 ·h -1 propofol protected cognitive function in aging rats with CH and being subjected to an ORIF surgery.
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. 1A).
Treatments with isoflurane or propofol alone were not able to prevent CA1 neuronal death in aging rats with cerebral hypoperfusion and being subjected to an ORIF surgery.
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 survival 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. 1 (Fig. 1

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 functional component of the neural substrate involved in cognitive functions. 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  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.

Discussion
In our study, aging (16-18 month) rats were chosen as the test subject. All the rats received ligation of the bilateral CCA to mimic the pathological process of CAS. 30 days after the ligation surgery, the ORIF surgery was operated and different anesthesia was given according to the group. After the ORIF surgery, behaviour experiments (FC test) were carried out to evaluate cognitive function of rats. Histological analyses (Nissl staining) were used to explore neuronal damage and biochemical analyses (western blotting) for the harvested rat brain tissues were performed to detect molecular changes.
The first thing that needs to be argued is the selection and intervention of the test subject. The incidence of PND in orthopedic patients varies from 16% to 45%, although it can be as high as 72% [29] and it has been proved that aging is a risk factor [30]. That's why we chose aging rats to be the test subject. CH has been reported to be one of the key factors in the development of cognitive impairment [31]. The underlying mechanism could be hypoxia-induced white matter damage, microvascular inflammation and neuro-gliovascular dysfunction [32]. We deem that aging patients with perioperative CH need more attention on the selection of surgery and anesthesia. Moreover, CAS detected in population older than 65 is 75% for men and 62% for women, with prevalence of stenosis ≥50% in this population 7% for men and 5% for women [33]. Taking incidence into account, we therefore used ligation of CAA induced CH aging rats in this study as the subject. Since it is difficult to separate clinical anesthesia and surgery, and our main purpose is to explore the combined effects of the two factors, no separate anesthesia group was set up in this study, which is consistent with most current studies [34-36].
FC test is a very sensitive and effort-independent test of learning and memory [37]. 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 [38]. After the ORIF surgery and anesthesia, the rats were placed in the same chamber as the one during the FC training phase. No tone was made during the rats were in the case. Under this circumstance, freezing behaviors rely on hippocampal memory. On the other hand, freezing behavior from rats exposed in the different chamber and given tone stimulus rely on hippocampal independent memory. In the current study, compared to group C, freezing time in group I, P and IP 2 all shortened significantly, while there was no obvious difference between group C and IP 1 . The only difference intervention between group I, P, IP 2 and IP 1 was anesthesia method. Our results suggest that hippocampal dependent memory was not 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 [39]. More than that, this area is vulnerable to ischemia injury [40]. Thus, we chose hippocampal CA1 area to measure the number of survival neurons and expression of certain protein. On the Day 1, numbers of neurons in group I decreased obviously compared to group C and on the Day 7 numbers of neurons in group I and P decreased obviously compared to group C. The difference between group C and combination anesthesia groups had not statistical significance. Thus, we can 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 [41]. 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 [41]. 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). The trend was coincident with the change of freezing time. 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 [42].
Expression of BiP and CHOP are evidence of heightened ER stress [11]. Prolonged or excess CHOP expression has been accepted as key to ER stress-related apoptosis [27]. In our study, 1% isoflurane and 20 mg·kg -1 ·h -1 propofol did not cause CHOP activation on neither one nor seven days after administration of anesthetics, while other ways of general anesthesia increased expression of CHOP variously. It suggests that these three ways of general anesthesia have caused severe or chronic ER stress that was far more beyond what UPR could buffer and led to cell apoptosis. BiP normally alleviates the UPR and is anti-apoptotic [26]. 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 UPR to transport unfolded/misfolded proteins to cytoplasm and trigger ERAD [43]. 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, like what group IP 1 did in this study. If not, prolonged or excess expression of CHOP will lead to apoptosis [44] , like what group I, P and IP 2 did in this study. Different anesthetic schedules alter neural substrate components (GABA A R α1 subunit) accounting for cognitive functions, even though in some cases the stimulation is not severe enough to cause apoptosis, it still may either aggravate (or induce), but also prevent, cognitive impairment. (Fig.5)

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 aging rats with CH during orthopedic surgery. The underlying mechanism of this phenomenon is related to alleviation of ER stress.

Methods
In our study, a ligation of bilateral CCA surgery [44] was adopted to prepare rats as CH The surgery was carried out with aseptic techniques. The left hind paw of surgical rats was shaved and disinfected with iodine tincture. After the skin was incised, an insertion of a 0.38 mm pin was performed in the intramedullary canal. Once the tibia was internally fixated, the bone was fractured in the middiaphysis (tibial, midshaft) using surgical pliers.
The skin was sutured with 8/0 Prolene sutures. Only skin incisions and sutures were performed on rats in group C. During the surgery, a heating lamp was used to help maintaining the body temperature of anesthetized rats at 37 ± 0.5°C. Postintervention rats were moved to heated pads for recovery and then delivered back to their own cage, where food and water were sufficient. For post-procedural pain relief, the rats were administered buprenorphine (0.05 mg/kg, subcutaneous) twice daily for 3 days [48].

Fear conditioning test
The fear conditioning (FC) test was carried on to estimate cognitive function [38]. The FC test consisted of a training phase at 24 h prior to ORIF operation and an evaluation phase on days 1 and 7 after ORIF when hippocampal-dependent memory was assessed.
During the training phase, rats were placed in a chamber (Ugo Basile, Italy) and allowed to adapt to the environment for 120 s. After adaption, they were stimulated by a 20 s 70-dB tone (conditional stimulus). Then there was an interval of 25 s. After the interval, rats were stimulated by a 0.70 mA electrical foot-shock for 2 s (unconditional stimulus). After six pairs of conditional-unconditional stimuli, the rats learned the association and established long-term memory. The pairs of conditional-unconditional stimuli were separated by 60 s inter-training intervals. Each training chamber was cleaned with 95% ethyl alcohol before the placement of a rat and was illuminated only with a 10 W bulb in a dark experimental room.
During the evaluation phase, rats were placed again in the training chamber for 5 min without tone and foot shock. Each animal's freezing behavior (without any movements) was scored by the ANY-maze video tracking system (Stoelting, Illinois, USA). The percentage of time in freezing behavior was calculated using the formula of 100*f/5min, where f was the total of freezing time in the 5 min. Freezing time measured during exposure to the known context, or after a conditional stimulus in the known context, reflects hippocampal-dependent memory whereas assessment during delivery of the conditional stimulus (tone) assesses hippocampal-independent memory [38]. Thus the results in this experiment were used to assess hippocampus-dependent memory.

Nissl staining
On days 1 and 7 after ORIF, rats (n=8/group) were first anaesthetized with 10% thiobutabarbital (100 ml/kg, i.p.). Perfusion with saline was given to the rats before the heart stops, followed by perfusion with 4% paraformaldehyde solution. Then the brain was taken out and fixed in 4% paraformaldehyde for 24 h.    7 after ORIF. Data was expressed as mean ± SD (n=8/group). Note that ORIF resulted in a significant increase of the number of expression of BiP in CA1 in IP2, I and P groups, which was prevented by the anesthetic schedule in the IP1 group.
* P < 0.05 compared with group C; # P < 0.05 compared with group IP1; ^ P < 0.05 compared with group IP2. propofol inhibited expression of CHOP while other groups did.