Abstract
Context: Hypotension causes histologic changes in the hippocampal CA1 area, while behavior remains unchanged. We believe that an even stronger insult may also cause behavioral changes.
Objective: We used a rat hemorrhagic shock model plus temporary hypoxia to assess functional outcome at different time points post-injury. Our hypothesis is that the damage can be attenuated by the use of isoflurane.
Materials and methods: Rats were subjected to brief hypotension. Animals were evaluated at different time points after receiving hypoxia and hypotension, with and without isoflurane treatment.
Results: The administration of isoflurane after the insult protected the animals from memory alterations. No histopatologic changes were found in any of the groups.
Discussion and conclusions: This observation suggests that in this model of hypotension plus hypoxia there is mild cerebral damage that is reflected by memory changes. Exposure to isoflurane after the insult can prevent the onset of memory deficits.
Introduction
Recent publications have shown that hypotension during surgery can produce a range of effects from postoperative memory deficitsCitation1–3 to an increase in mortality during the next year after surgery; specially in elderly patientsCitation4. Permanent, stable and adequate blood flow to the brain is necessary during general anesthesia in order to assure safe recovery and normal brain function after a surgical interventionCitation5,Citation6.
Some researchers have demonstrated that hypotension can have deleterious effects on brain histology. Yamauchi et alCitation7,Citation8. described progressive damage to several regions of the brain after three separate 1-min episodes of hypotension (mean arterial pressure 25 mmHg) after 7 days of recovery in rats. Although postoperative cognitive dysfunction (POCD) does not have a clear etiology, some physicians believe intraoperative hypotension (IOH) may be responsibleCitation9,Citation10. Schutz et alCitation11. found that a 30-min period of hypotension does not have any effect in structural and motor deficits but has some effects in recovering cognition. Hypertensive patients seem to be more sensitive to IOH: Yocum et alCitation12. found a clear relation between IOH in hypertensive patients and decline in cognitive function. Patients with advanced age seem more vulnerable to POCD after IOHCitation13–15 but not all the studies have shown a clear relation between IOH and POCDCitation16,Citation17.
In our laboratory, using a rat model of hypotension, we have found a clear relation between IOH and histologic changes in the hippocampus but the cortex and memory were not affectedCitation1.
Animal models of hypotension and hypotension plus hypoxia are necessary to study the effect of these variables and their effects in the central nervous system. In this study, we evaluated the effect of a short period of profound hypotension plus hypoxia. Histology and behavioral testing was used in order to clearly understand the relation between these variables.
Materials and methods
Regulations
The present study was approved by the division of Comparative Medicine at the University of South Florida (USF). The experiments were done in following the guidelines of the IACUC of the University of South Florida’s College of Medicine.
Five groups of animals were used in this study; the first four groups shown were designed to characterize the damage caused by hypotension plus hypoxia and the isoflurane group was added to determine if the expected damage could be reversed with this anesthetic. The animals were randomly assigned to the groups. We use a computer generated code to randomly distribute them into the different groups.
The creation of new memories after the insult was evaluated using the passive avoidance paradigm as described by Saporta et alCitation18. A forty-eight point scale was used for neurologic assessment at different time points in a 2-week periodCitation2.
Animals
Male Sprague Dawley rats from Harlan Laboratories (Indianapolis, IN), with ages between 60 and 90 days and weights between 250 and 350 g were used for this experiment. Upon arrival to the USF College of Medicine Vivarium, the rats were housed in a climate-controlled room in acrylic cages in groups of two with free access to water and food and were left in quarantine for at least a week before the experiment took place.
Groups
The animals were divided in five groups ():
Shams. Control group, these animals received no hypotension.
These animals received 3 min of hypotension plus hypoxia and were euthanized 24 h after the surgery.
These animals received 3 min of hypotension plus hypoxia and were euthanized 4 days after the surgery.
These animals received 3 min of hypotension plus hypoxia and were euthanized 14 days after the surgery.
These animals received 3 min of hypotension plus hypoxia, they also received treatment with isoflurane per 90 min after the insult and were euthanized 14 days after the surgery. The complete time course of the experiments is summarized in .
Table 1. Study design.
Table 2. Time course.
Neurologic assessment
Neurologic evaluation was performed twice in every animal. The first evaluation was the day before the surgery; this test was done to assure that neurologically the rats were intact. Having an animal with any kind of neurologic deficit would prevent it to be part of the study. No animals were withdrawn from the study for this reason. The second evaluation was performed before tissue collection. We used a forty-eight point neurologic score to evaluate the animals. This scale was taken from Yokoo et alCitation2, it is the result of combining elements of several systems; 0 points indicate normal function, a total of 48 deficit point is possible ().
Table 3. Forty-eight points neurological score.
Passive avoidance
Memory was assessed using the passive avoidance paradigm (PA); habituation, training and testing were done after the hypotensive/hypoxic insult. Habituation occurred 24 h after surgery: during habituation the animal was placed in a plexiglass box, the floor of the cage is an exposed metallic grid that can be manually electrified; on habituation day the animal is placed on a platform that prevent the animal from touching the grid and animals tend to spontaneously step down the platform and the latency is recorded. On the next day the grid is electrified with 0.5 mA and again we measured the time they remained in the platform before they step down of it; once the animal have placed the four limbs down the platform, the animal received an electric shock (0.5 mA) for 3 s. If memory process is intact the animal learns that the safe place to be is the platform. A Day 3, 4, 7 and 14 we placed the animals on the platform and measured the time the animal took to step down of it, with a maximum of 5 min (300 s). Latencies greater than 300 s were assigned this value. Finally on tissue collection day we recorded the time the animal remained on the platform up to 5 min. PA was only measured in the control group and the group euthanized at Day 14Citation18. The forty-eight point neurologic exam and the passive avoidance paradigm were done by a researcher blinded to the groups.
Anesthesia
After weight was recorded, the animal was anesthetized in an induction chamber with 5% isoflurane in oxygen. After the rat was fully anesthetized we changed the rat from the induction chamber to a mask with 1–2% isoflurane in oxygen. Rats remained anesthetized at all times. Animals in the isoflurane group received additionally 1% isoflurane in oxygen by mask for 90 min after the insult.
Surgery
After shaving the neck, the area was cleaned with iodine and a medial linear incision was made. Plastic catheters were inserted in the jugular vein and both carotid arteries (bilateral). Arterial lines were used for blood pressure and blood suction; the venous line was used for blood reinfusion. Blood was aspirated until the mean arterial pressure (MAP) reached a point below 30 mmHg. Hypoxia was induced with a mixture of 12% oxygen and 88% nitrogen, the animal breathed the mixture for 5 min before the hypotensive period and during the hypotensive period. As soon as MAP was below 30 mmHg, the chronometer was set for 180 s. Between 8 and 15 cc of blood were taken to reach the MAP of 30 mmHg or less. After the hypotensive episode, the blood was reinfused using the venous catheter, and the hypoxic mixture was stopped and 100% oxygen was given to the animal until the end of the surgery. The catheters were removed and the wound closed with a skin stapler. During the procedure the animals were placed on a heated pad to prevent hypothermia. Recovery from procedure was in a fresh cage. Sham animals received the same procedure as the other animal except for hypotension and hypoxia. The animals were euthanized at Day 14 and were only compare with the treated animals euthanized at Day 14.
Physiologic parameters
Physiologic parameters were measured to assure that the results we obtained were due to the insult and could not relate to alterations in physiologic changes. The variables we measured were: weight, temperature (taken with a rectal thermometer) and maintained using a heated pad, hemoglobin saturation (measured with a pulse oximeter), heart rate and blood pressure (measured directly using SurgiVet Advisor Monitor, Model No. 92V303100, Smiths Medical, Dublin, OH). Readings were taken before, during and after the ischemic event. Weight was measured before the surgery and at euthanasia.
Brain extraction and sectioning
The animals were euthanatized with an overdose of CO2. Immediately after, the animal was perfused with normal saline solution 0.9% followed by 4% paraformaldehyde. The brains were harvested, stored in plastic tubes with 4% paraformaldehyde for 24 h, and then changed to 10, 20 and 30% sucrose every 24 h. Brains were then sectioned at 40 μm with cryostat HM 550 from MICROM International GmbH (Walldorf, Germany), the chamber temperature was turned down to −22 °C. A series of five sections spaced ∼960 microns apart were stained. NeuN and Nissl stains were used for histologic analysis. Sections were mounted for histopathology analysis and the remaining sections were stored in phosphate-buffered saline plus azide.
NeuN
NeuN (Neuronal Nuclei) is used to label neurons. Immunoreactivity is observed after mitosis. NeuN is primarily localized in the neuronal nuclei with lighter staining in the cytoplasm. The staining was done following the instructions from the manufacturer (Chemicon, EMD Millipore Corporation, Billerica, MA).
Nissl
Nissl is used for detection of Nissl bodies in the cytoplasm of Cells. The Nissl body stain purple-blue. We used this staining to count the number of cells in the Hippocampus.
Statistical analysis
Data is presented as mean ± SD. To count the number of NeuN positive cells and Nissl cells, we used unbiased stereology (Optical Fractionator, MicroBrightField, Williston, VT) as described in other publicationsCitation19–21. For this purpose the Stereologer from Stereology Resource Center, Chester, MD was used. The results are an estimate of the total number of cells. Neurologic score and passive avoidance data were also evaluated. The results were analyzed using GraphPad Prism 5.0 for Mac (GraphPad Software, Inc., La Jolla, CA). One-way analysis of variance (ANOVA) was used followed by Bonferroni’s multiple comparison test for NeuN and Nissl. For neurologic evaluation, we used Dunnett’s multiple comparison test after ANOVA. Passive avoidance was analyzed using the Mixed Effect Model to study the general differences between groups during the 2-week period; for specific time points, we used ANOVA followed by Bonferroni’s multiple comparison test. p Value of <0.05 were considered statistically significant.
Results
We designed the experiment with 50 animals, 10 of them died during or after surgery (two per group). Necropsies showed that pulmonary embolism, bleeding from the arterial incision and neurologic deficits were the cause, these animals were not replaced and the experiment was completed with the remaining animals.
Physiologic variables are presented on . The MAP combining all the injured groups before the insult was 110 mmHg, during the insult the MAP was 27 mmHg and after the blood was reinfused the MAP was 87 mmHg. We did not find any statistically significant differences between the preoperative values and the postoperative values. Normal animals with no injury, gained an average of 24 g in 2 weeks, the animals that received the insult gained an average of 25 g in the same period; the animals that received the insult plus were treated with isoflurane 90 min after the insult, gained 8 g in average. Statistically we did not find any significant difference. The animals that received the insult have lower weight at Day 1 and 4 after the injury but by Day 14 the weight was comparable with the control animals. The mean hemoglobin saturation during the episode measured immediately before the hypotensive insult was 74.2; the values before and after the insult were 94.8 and 95.5, respectively. No significant changes in temperature and heart rate were found.
Table 4. Physiological variables.
Neurologic performance was evaluated in all animals and assigned a value in a neurologic score (). Behavior was evaluated on postoperative Day 1, 4 and 14. Only the groups of rats that survived 14 days were evaluated. Group 1 has all the animals with the sham surgery that received no insult; Group 2 has the animals that received the insult and were euthanized at Day 14 and Group 3 is the group that received the insult and also was exposed to isoflurane for 90 min. No statistically significant changes were found (p > 0.05). It is important to mention that some animals also showed some degree of paralysis after the surgery but by the time of the neurologic evaluation it was no longer present.
Figure 1. Neurologic score. The figure shows small and non-significant changes in the neurologic score. Only the rats euthanized at Day 14 were subject of neurologic evaluation.
Passive avoidance as described by Saporta et al.Citation18, was used to test the ability to recall new memories. There was a statistically significant difference in memory during the 2-week period. This difference was more evident at Day 7 and persisted at Day 14 demonstrating that profound hypotension plus hypoxia for short periods of time can impair the creation of new memories. Animals treated with isoflurane after the insult did no show any memory impairment ().
Figure 2. Passive avoidance. There is a statistically significant difference between Groups 1 and 2 and 2 and 3. There is no statistically significant difference between Groups 1 and 3.
Stereology was used to estimate the total number of positive NeuN cells in the CA1 area of the hippocampus. No difference in cell numbers were seen between control rats and rats that received 3 min of profound hypotension plus hypoxia at any time point (p > 0.05, ).
Figure 3. NeuN positive cells in animals subject to 3 min of hypotension/hypoxia. No statistically significant changes were found. Similar number of cells were found at all the time points analyzed.
Nissl stained cells in the CA1 area of the hippocampus were counted and although we found a pattern of decrease in the number of cells (Day 14), there were no statistically significant changes at any time point after the hypotension plus hypoxia insult ().
Figure 4. Nissl cells in CA1. No significant changes in the number of cells in the CA1 area of the hippocampus were found.
Discussion and conclusions
Although this model of insult is a temporary model (3 min of hypotension plus 8 min of hypoxia), we occluded the two common carotid arteries making it a model of constant hypoperfusion or global ischemia. However, even with this permanent ischemia, we did not find changes in the CA1 area.
Weight is always a good indicative of general conditions, normal animals increase in weight day after day; animals that received the insult lost a statistically significant amount of weight after the insult but by Day 14 this difference vanish among the groups.
Oxygen saturation was recorded with a pulse oximeter and maintained at an average of 74.2 ± 6.6% assuring that, although we used a facial mask for administration, the mixture of 12% oxygen plus 88% nitrogen was effective in diminishing the saturation levels.
Between 8 and 15 ml of blood were taken from the animal to decrease the blood pressure to a mean of 27.1 ± 5.4 mmHg. In previous experiments, we have used lower MAP (<20 mmHg)Citation22 but in a preliminary study using that hypotension plus hypoxia the mortality was too high, for this reason we maintained a higher MAP (MAP < 30 mmHg).
Temperature is a recognized neuroprotectant; the results showed that the heated pad is a good device to control this variable. The temperature at the beginning of the surgery was 95.5 ± 2.9 and 93.5 ± 1.6 at the end of the surgery.
Neurologic scale evaluation showed some negative effects related to the procedure but statistically results were not significant. Palpebral ptosis was the main motor alteration we found. In humans, the dissection of the internal carotid artery, typically manifests as an oculosympathetic palsy (myosis and palpebral ptosis)Citation23.
Passive avoidance was used to test anterograde memories and a negative effect was seen 1 week after the insult. Memory deficit persisted 2 weeks after the insult. In a recent article, Bekker et alCitation3. found impairment after hypotension in long-term associative memory. Although we did not use the same model of hypotension that he didCitation24, and we also added hypoxia, the present study may highlight a similar mechanism. The memory deficit was reversed by the use of isoflurane per 90 min after the insult.
Schutz et alCitation11. have demonstrated that hemorrhagic hypotension does not cause structural damages but can cause cognitive alterations. Our study showed a similar result, we found long-term (2 weeks) memory impairment without neurologic changes or histologic changes in the hippocampus.
The functional improvement because of isoflurane was not unexpected, given the previously published articles describing improved neurologic outcome and neuroprotection furnished by isofluraneCitation25–27. The responsible mechanisms included decreased sympathetic activityCitation25 and protection from adverse effects such as apoptosis, degeneration, inflammation and energy failure which are involved in causing neuronal damage in chronic degenerative diseases, ischemia, stroke or nervous system traumaCitation26. Li et alCitation28. recently reported that isoflurane administration reduced brain infarct volumes, apoptotic cells in the ischemic penumbral brain tissues and neurologic deficits in rats after 4 weeks from ischemic brain damage. They also found that the neuroprotective effects were associated with decreased proinflammatory cytokines. Neuroprotection may be found.
We performed our experiments on male animals only to avoid possible effects of the estrous cycle on the resultsCitation29.
Heart rate values reported in indicated that the animals were in a general good physiologic condition. In fact, heart rate values we measured are similar to those previously reported for the healthy rats in the literatureCitation30,Citation31.
Thus, on the basis of recent work, an organic substrate for the isoflurane induced.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References
- Chaparro RE, Quiroga C, Bosco G, et al. Hippocampal cellular loss after brief hypotension. SpringerPlus 2013;2:23 . doi:10.1186/2193-1801-2-23
- Yokoo N, Sheng H, Mixco J, et al. Intraischemic nitrous oxide alters neither neurologic nor histologic outcome: a comparison with dizocilpine. Anesth Analg 2004;99:896–903
- Bekker A, Haile M, Li YS, et al. Nimodipine prevents memory impairment caused by nitroglycerin-induced hypotension in adult mice. Anesth Analg 2009;109:1943–8
- Bijker JB, van Klei WA, Vergouwe Y, et al. Intraoperative hypotension and 1 year mortality after non cardiac surgery. Anesthesiology 2009;111:1217–26
- Rubio A, Hakami L, Munch F, et al. Noninvasive control of adequate cerebral oxygenation during low-flow antegrade selective cerebral perfusion on adults and infants in the aortic arch surgery. J Card Surg 2008;23:474–9
- Moritz A, Koci G, Steinlechner B, et al. Contralateral stroke during carotid endarterectomy due to abnormalities in the circle of Willis. Wien Klin Wochenschr 2007;119:669–73
- Yamauchi Y, Kato H, Kogure K. Brain damage in a new hemorrhagic shock model in the rat using long-term recovery. J Cereb Blood Flow Metab 1990;10:207–12
- Yamauchi Y, Kato H, Kogure K. Hippocampal damage following repeated brief hypotensive episodes in the rat. J Cereb Blood Flow Metab 1991;11:974–8
- Duschek S, Schandry R. Reduced brain perfusion and cognitive performance due to constitutional hypotension. Clin Auton Res 2007;17:69–76
- Wharton W, Hirshman E, Merritt P, et al. Lower blood pressure correlates with poorer performance on visuospatial attention tasks in younger individuals. Biol Psychol 2006;73:227–34
- Schutz C, Stover JF, Thompson HJ, et al. Acute, transient hemorrhagic hypotension does not aggravate structural damage or neurologic motor deficits but delays the long-term cognitive recovery following mild to moderate traumatic brain injury. Crit Care Med 2006;34:492–501
- Yocum GT, Gaudet JG, Teverbaugh LA, et al. Neurocognitive performance in hypertensive patients after spine surgery. Anesthesiology 2009;110:254–61
- Zuccala G, Onder G, Pedone C, et al. Hypotension and cognitive impairment: selective association in patients with heart failure. Neurology 2001;57:1986–92
- Yap PL, Niti M, Yap KB, Ng TP. Orthostatic hypotension, hypotension and cognitive status: early comorbid markers of primary dementia? Dement Geriatr Cogn Disord 2008;26:239–46
- Qiu C, Winblad B, Fratiglioni L. The age-dependent relation of blood pressure to cognitive function and dementia. Lancet Neurol 2005;4:487–99
- Williams-Russo P, Sharrock NE, Mattis S, et al. Randomized trial of hypotensive epidural anesthesia in older adults. Anesthesiology 1999;91:926–35
- Moller JT, Cluitmans P, Rasmussen LS, et al. Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post-Operative Cognitive Dysfunction. Lancet 1998;351:857–61
- Saporta S, Borlongan CV, Sanberg PR. Neural transplantation of human neuroteratocarcinoma (hNT) neurons into ischemic rats. A quantitative dose-response analysis of cell survival and behavioral recovery. Neuroscience 1999;91:519–25
- Shi L, Molina DP, Robbins ME, et al. Hippocampal neuron number is unchanged 1 year after fractionated whole-brain irradiation at middle age. Int J Radiat Oncol Biol Phys 2008;71:526–32
- Fitting S, Booze RM, Hasselrot U, Mactutus CF. Dose-dependent long-term effects of Tat in the rat hippocampal formation: a design-based stereological study. Hippocampus 2010;20:469–80
- Skoglund TS, Pascher R, Berthold CH. Aspects of the quantitative analysis of neurons in the cerebral cortex. J Neurosci Methods 1996;70:201–10
- Rafael E, Chaparro MD, Carolina E, et al. Brief systemic hypotension results in cortical neuronal loss in Sprague Dawley rats. Orlando: American Society of Anesthesiology; 2008
- Eschmann L, Favrat B, Botez S, Wuerzner K. Partial Horner's syndrome and facial pain: a diagnosis one should not miss. Rev Med Suisse 2006;2:544–6
- Chaparro E, Erasso D, Quiroga C, et al. Repetitive intraperitoneal caspase-3 inhibitor and anesthesia reduces neuronal damage. J Enzyme Inhib Med Chem 2013;28:1324–30
- Engelhard K, Werner C, Reeker W, et al. Desflurane and isoflurane improve neurological outcome after incomplete cerebral ischaemia in rats. Br J Anaesth 1999;83:415–21
- Schifilliti D, Grasso G, Conti A, Fodale V. Anaesthetic-related neuroprotection: intravenous or inhalational agents? CNS Drugs 2010;24:893–907
- Gigante PR, Appelboom G, Hwang BY, et al. Isoflurane preconditioning affords functional neuroprotection in a murine model of intracerebral hemorrhage. Acta Neurochir Suppl 2011;111:141–4
- Li L, Peng L, Zuo Z. Isoflurane preconditioning increase B cell lymphoma 2 expression and reduces cytochrome C release from the mitocondria in the ischemic penumbra of rat brain. Eur J Pharmacol 2008;596:106–13
- Rubini A, Bondì M. Effect of the oestral cycle on respiratory mechanics in the rat. Acta Physiologica 2007;189:379–83
- Rubini A, Carniel EL, Natali AN. The effect of body warming on respiratory system stress recovery in the rat. Acta Bioeng Biomech 2012;14:59–66
- Rubini A, Bosco G. The effect of body warming on the dynamic respiratory system compliance-frequency of breathing relationship in the rat. J Biol Phys 2013;39:411–18