Hippocampal cellular loss after brief hypotension

  • Rafael E Chaparro1, 2,

    Affiliated with

    • Carolina Quiroga1, 2,

      Affiliated with

      • Gerardo Bosco4Email author,

        Affiliated with

        • Diana Erasso2,

          Affiliated with

          • Alessandro Rubini4,

            Affiliated with

            • Devanand Mangar3,

              Affiliated with

              • Andrea Parmagnani4 and

                Affiliated with

                • Enrico M Camporesi1

                  Affiliated with


                  DOI: 10.1186/2193-1801-2-23

                  Received: 19 October 2012

                  Accepted: 23 January 2013

                  Published: 25 January 2013


                  Brief episodes of hypotension have been shown to cause acute brain damage inanimal models. We used a rat hemorrhagic shock model to assess functionaloutcome and to measure the relative neuronal damage at 1, 4 and 14 dayspost-injury (3 min of hypotension). All rats underwent a neurological assessmentincluding motor abilities, sensory system evaluation and retrograde memory atpost-hypotensive insult. Brains were harvested and stained for Fluorojade C andNissl. Stereology was used to analyze Fluorojade C and Nissl stained brainsections to quantitatively detect neuronal damage after the hypotensive insult.Statistical analysis was performed using Graphpad Prism 5 with the Bonferronitest at a 95% confidence interval after ANOVA. A Mixed Effect Model was usedfor the passive avoidance evaluation. Stereologically counted fluorojadepositive cells in the hippocampus revealed significant differences in neuronalcell injury between control rats and rats that received 3 min of hypotension oneday after insult. Quantification of Nissl positive neuronal cells showed asignificant decrease in the number hippocampal cells at day 14. No changes infrontal cortical cells were evident at any time, no significative changes inneurological assessments as well. Our observations show that brief periods ofhemorrhage-induced hypotension actually result in neuronal cell damage inSprague–Dawley rats even if the extent of neuronal damage that wasincurred was not significant enough to cause changes in motor or sensorybehavior.


                  Hypotension Rats Hemorrhagic shock model Neuronal damage


                  Maintenance of adequate blood flow to the brain is necessary in the course of generalanesthesia in order to assure safe recovery and normal brain function after surgicalintervention (Rubio et al. 2008; Moritz et al. 2007). Predictable models of neuronal loss after progressive low bloodpressure insults have been developed (Yamauchi et al. 19901991) For instance, Yamauchi and colleagues describedselective progressive damage to regions of the brain after two or three minuteepisodes of profound hypotension (low blood pressure, 25 mmHg) one week after thehypotensive insult (Yamauchi et al. 19901991). These changes were attributed to neuronal necrosis(Fukuda and Warner 2007). However, this study did not measurefunctional behavior after recovery from hypotension. Cognitive dysfunction has beenlinked with hypotension (Duschek et al. 2007 Wharton et al.2006), especially in elderly patients (Zuccala et al.2001; Qiu et al. 2005). Severalstudies have studied the relationship between hypotension during surgery andneurological performance after surgery but so far a clear link has not been found(Williams-Russo et al. 1999). The largest study thatevaluated this matter was “The International Study of Postoperative CognitiveDysfunction” but no association between surgical blood pressure andpostoperative cognitive function was found (Moller et al. 1998). In contrast, other researchers have found a link betweenpostoperative cognitive function and blood pressure during surgery (Schutz et al.2006; Yocum et al. 2009).

                  With this in mind we designed an animal study to test the hypothesis that hypotensiveepisodes during surgery may cause long-term functional alterations. A secondaryobjective of this study was to characterize an animal model of hypotension thatwould produce consistent cerebral damage that could be used for future analyses.

                  Materials and methods


                  This study was approved by the division of Comparative Medicine at the Universityof South Florida (USF). All experiments were done following IACUC guidelines.Male Sprague Dawley rats were separated into four groups as follows; groups 1, 2and 3 received 3 min of hypotension - 1 min every hour - and were evaluated at1, 4 and 14 days, respectively. An additional group of rats, group 4, received asham operation (Table 1).
                  Table 1

                  Physiological variables


                  Group 1

                  Group 2

                  Group 3


                  Euthanized at day 1

                  Euthanized at day 4

                  Euthanized at day 14


                  (n = 7)

                  (n = 6)

                  (n = 5)

                  Initial weight (g)

                  352 ± 11

                  334 ± 12

                  344 ± 22

                  Final weight (g)

                  349 ± 13

                  362 ± 29

                  380 ± 39

                  Initial temp (°F)

                  94.8 ± 2.2

                  93.02 ± 2.6

                  92.58 ± 4.3

                  Final Temp (°F)

                  89.6 ± 7.5

                  91.88 ± 2.6

                  88.8 ± 3.1

                  First MAP (mmHg)

                  19 ± 2

                  20 ± 2

                  19 ± 2

                  Second MAP (mmHg)

                  19 ± 1

                  19 ± 1

                  20 ± 1

                  Third MAP (mmHg)

                  20 ± 2

                  18 ± 1

                  19 ± 1

                  Initial HR (bpm)

                  254 ± 49

                  271 ± 50

                  248 ± 61

                  Final HR (bpm)

                  267 ± 49

                  233 ± 59

                  250 ± 54

                  Initail Hb. Saturation

                  97 ± 2

                  94 ± 6

                  95 ± 4

                  Final Hb. Saturation

                  95 ± 5

                  94 ± 4

                  98 ± 7

                  Values are mean ± SE. MAP = Mean Arterial Pressure, HR = Heartrate, #To = Temperature, BPM = Beats Per Minute,Hb = Hemoglobin, F° = Fahrenheit. No significantdifferences were found.


                  Male Sprague Dawley rats aged between 60 and 90 days with weights between 250 and350g from Harlan Laboratories (Indianapolis, IN) were used. Upon arrival to theUSF College of Medicine Vivarium, rats were housed in a climate controlled roomin plastic cages in groups of two with free access to water and food and wereleft in quarantine for one week before the experiment took place.

                  Neurological assessment

                  The Neurological state of the rat was assessed using the forty-eight point scale(Yokoo et al. 2004). The test was done at 2 differenttime points. The first evaluation was the day before surgery to assure that allrats were neurologically intact. Any rat that demonstrated a neurologicaldeficit was not used for the study. No rats were withdrawn from the study underthose criteria. The second evaluation was completed before tissue collection.The passive avoidance test was used in order to evaluate memory functionfollowing hypotension as described by Saporta (1999).Habituation is the first step. The animal was placed on a platform in aplexiglass box. The amount of time that the rat remained on the platform beforethey stepped down was measured. The following day, the rats were placed on theplatform and received an electric shock (0.5 mA) for three seconds if theystepped down from the platform. After habituation and training, the rats learnedto remain on the platform to avoid being shocked. All rats were trained beforethe hypotensive insult and were tested on the day before and the day ofeuthanasia. For all instances, the animal was placed on the platform and time tostep-down was measured for a maximum of 5 min (300 seconds). Latencies greaterthan 300 seconds were assigned a default value of 300 seconds.


                  After the weight was recorded, the animal was anesthetized in an inductionchamber with 5% Isoflurane in 100% Oxygen. After the rat was fullyanesthetized we changed the animal from the induction chamber to a mask with 1to 2% Isoflurane in 100% oxygen. The rats were continuously anesthetizedduring the three episodes of hypotension.


                  After shaving the neck, the area was cleaned with iodine and a medial linearincision was made. Plastic catheters were inserted in the jugular vein and bothcarotid arteries. The arterial lines were used for blood pressure quantificationand blood aspiration. The venous line was used for blood reinfusion. The bloodwas rapidly aspirated until the mean arterial pressure (MAP) reached a point ator below 20 mmHg. The blood was withdrawn and reinfuse is between 10 to 20seconds and the amount range was between 8 to 15 cc. The goal was not to reducethe circulating blood in an specific amount but achieve a blood pressure closeto 20 mmHG. If the animal did not reach a MAP below 20 at any of the 3 episodes;it was excluded from the experiment. When MAP was below 20, the chronometer wasset for 60 seconds. Between 8 cc to 15 cc of blood were taken to reach MAP of 20or less. After the minute, the blood was reinfused using the venous catheter.During the procedure the rats were placed on a heated pad to preventhypothermia. At the end of the third hypotensive episode the catheters wereremoved and skin was closed with a skin stapler. The rats recovered in a cleancage.

                  Physiological parameters

                  The following variables were measured: Weight, temperature (measured with arectal thermometer), hemoglobin saturation (Hb Sat), measured with a pulseoximeter, heart rate (HR), and blood pressure (BP) (obtained directly from anarterial catheter placed in the carotid artery) (SurgiVet Advisor Monitor, modelnumber 92V303100 was used for Hb Sat, HR and BP). Readings were made before andafter each ischemic event. Weight was measured right before the surgery and ateuthanasia.

                  Brain extraction and sectioning

                  The rats were euthanatized with an overdose of CO2 and the arterialtree 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 hours, and then changed to 10%, 20% and 30%sucrose every 24 hours. The brains were sectioned at 40 μm with cryostat HM550 from MICROM International, at a chamber temperature set at −22°C.A series of 5 coronal sections spaced approximately 960 microns apart weremounted for histopathology analysis and stained with Fluorojade C and Nissl. Thebrains were sectioned at 40 μm, the consecutive sections were collected ina 24 well plate previously fill out with PBS + azyde. The sectionscollected from wells # 1 and 13 were stain and mount on glass slides forcounting. We used stereology (optical dissector) to count cells in frontalcortex and CA1 area of hypocampus.

                  Fluoro jade C

                  Fluoro Jade C stain is commonly used in ischemia research to label degeneratingneurons regardless of the insult. Fluoro Jade C analysis was used to quantifythe number of degenerating cells in the cortex and CA1 area of the hippocampus;the method used has been described in detail (Ajmo et al. 2008). The sections were mounted on slides and air-dryed overnight;then, they were dipped in absolute ethyl alcohol 3 times, and 1 min in 70%ethyl alcohol and washed in running tap water 1 min, then stained with potassiumpermanganate to oxidize tissue for 15 min while shaking gently. 120 mg ofpotassium permanganate (KMnO4) 0.06% was diluted in 200 mL ofPBS. At this point the slides were protected from light. Staining with FluoroJade C 0.001% for 30 min was followed by 3 changes of water for 1 min andair-dry overnight. The next day under the fume hood, they were cleared in xylenefor 2 min, 3 times. Coverslip with DPX directly from xylene. Fluoro-Jade stockwas made with 0.01% Fluoro Jade C in water (dilute 2 mg/ 20 ml). The workingsolution was made with 0.001% Fluoro-Jade C in 0.1% acetic acid, afterdiluting 20 ml stock with 180 mL of water plus 200 uL of acetic acid.


                  Nissl is a classic stain used for detection of Nissl bodies in the cytoplasm ofcells, which will be stained purple-blue. Sections were mounted and air-dried ona slide warmer overnight. The following day the slides were dehydrated through100% and 95% alcohol to distilled water. The slides were stained in0.1% cresyl violet solution for 5 min and then rinsed with distilled water.The slides were then immersed in 95% ethyl alcohol for 30 min and posteriordehydrated in 100% alcohol 2 times for 5 min. Under the fume hood they werecleared in xylene for 2 min, 3 times and then immediately coverslipped withDPX.

                  Statistical analysis

                  The data are presented as Mean ± SE. Counting of Fluoro Jade Cpositive cells and Nissl cells was completed using unbiased stereology (Opticalfractionator) as described in by Mouton (2002). For thispurpose a Stereologer from Stereology Resource Center, Chester, Maryland wasused. The results are an estimate of the total number of cells. Neurologicalscore data were also evaluated. One-way Analysis of Variance was followed byBonferroni Multiple Comparison test. The results were analyzed using GraphPadPrism 5.0 for Mac. Passive avoidance was evaluated using a Mixed Effect Model. Ap-value of <0.05 was considered statistically significant.

                  Results and discussion

                  We designed the experiment with 40 rats, 7 of which died during surgery. Dead ratswere not replaced (See Table 1). Physiological variables wereconsidered and recorded. The baseline mean arterial pressure combining all thegroups was 100.3 ± 42 mmHg, 94.3 ± 38 mmHg beforethe second insult and 102 ± 40 mmHg before the last insult. Afterthe insult and blood re-infusion the mean arterial pressure was87.7 ± 56.2 mmHg, 94.5 ± 57.2 mmHg and92.7 ± 43.2 mmHg respectively. During the hypotension period, theblood was withdrawn until the MAP reach 20 or less. No statistically significantdifferences were found between the pre-operative values and the post-operativevalues. We also compared the weight before the ischemic event and at day 14 and didnot find any statistically significant change (Table 1). Inpilot studies we have tried 1 or 2 separate minutes of deep hypotension (MAP of 20or less). The first insult that was successful in finding cellular lost was withthree separate minutes of deep hypotension. We believe that accumulation of calciumis a key factor in developing the injury. Future experiments should focus oncounting calcium levels after different number of 1 min insults to corroborate thishypothesis.

                  Neurological performance was evaluated in all rats. Behavior measurements onpostoperative day 1, 4 and 14 were compared with control; at day one there werepositive findings (unilateral palpebral ptosis). These findings were not present atday 4 or 14. Although there was some neurological impairment 24 hours after theinjury, these changes were not statistically significant. Some rats showed somedegree of paralysis immediately after the surgery but recovered by the time of theneurological evaluation. 10% of the animals showed temporary paralysis of thelower extremities, affected animals recovered in the next 24 hours after surgery. Webelieve that spinal cord ischemia could be the etiology of this finding. One of theanimal models of spinal cord injury is achieved by temporal clipping of the thoracicaorta. We believe that the temporal paralysis that we have seen in some of thisanimals could be related to spinal cord ischemia.

                  Passive avoidance as described by Saporta (1999), was used totest the ability to recall old memories. During the habituation and training, ratslearned to stay on the platform to avoid an electric shock (Figure 1).
                  Figure 1

                  All the animals only received one electric shock at day 2 before theinjury. There is no significant differences between control animalsand ischemic animals at day 1, 4 or 14. Number of animals:total = 40; control = 10 animals; animals euthanizedat day 1 = 7 alive (3 died); animals euthanized at day4 = 6 alive (4 died); animals euthanized at day14 = 5 alive (5 died). Total number of deadanimals = 12.

                  We did not find any memory impairment in the rats after the surgery in comparisonwith the control rats.

                  Stereology was used to estimate the total number of positive Fluoro Jade C cells inthe frontal cortex and CA1 area of the hippocampus. No significant differences incellular injury represented by numerous fluoro jade positive cortical cells wereseen between control rats and rats that received 3 min of hypotension(p > 0.05). The pictures from Figure 2shown the groups represented in Figure 3B. The first oneis from an animal that received the sham operation, it shows a low number of fluorojade positive cells, in contrast, 24 hours after the insult there is an increase inthe number of cells that are positive for fluoro jade c (positive cells are greenand bright like the one shown with the arrow). This means that since the cells aredamage the stain was able to get into the genetic material. These changes are notnoticeable at day 4 or 14. This can be explained because at this late time pointsthe cells resolved the damage so they are either dead or they recovered from theinjury. For this reason it was important to check for the number of live cells atthis time points. We found congruent results. At day 1 and 4th the number of nisslcells are the same as normal animals but at day 14th the cells have died and thenumber of viable cells has decrease significantly.
                  Figure 2

                  Fluoro Jade C positive cells in Hypocampal CA1 area in rats subjected to3 non consecutive minutes of hypotension. Fluoro-Jade C positivecells in Hypocampal CA1 area in rats subjected to 3 non-consecutive minutesof hypotension. Fluoro-Jade C positive cells were absent in control(A), present at Day 1 after hypotension (B, arrow), andagain absent at Day 4 (C) and Day 14 (D) afterhypotension.

                  Figure 3

                  FluroJade C positive labeled in the (A) cortex and (B) CA1 region, incontrol group, 1, 4 and 14 days following hypotension. FluroJade Cpositive labeled was statistically significantly higher in the CA1 region ofrats one day after hypotension as compared to control rats*p < 0.05. The fire shows the results from staining thebrains with Fluoro Jade C. Panea A shows cell counting in frontalcortex. There is no significant difference in the number of Fluoro Jade Cpositive cells. This suggest that the cortex is not affected by the insult.Panel B shows cell counting in the CA1 area of the hyppocampus. Thenumber of fluoro jade C positive cells increase significantly*(p < 0.05) one day after the injury in comparison withnormal animals. This increase is not present at day 4 or 14th after theinsult. This increase represents cell damage, by day 4 or 14th the cellshave decided their fate and have survive of died. In either case they arenot fluorescent anymore.

                  On the other hand significant differences in cellular injury were seen betweencontrol rats and rats that received 3 min of hypotension on day one after the insultrepresented by numerous fluoro-jade C positive cells in the CA1 area of thehippocampus (p < 0.05). Figures 34. The Nissl stain, stains cells that haveintact genetic materials by the time they where fixed, we can interpret that as"live cells". Fluoro jade C is a stain meant to stain broken genetic material atfixation time, this cells are either dead or degenerating. The tissue collection wasdone at 3 different time points. 1, 4 and 14 days. The cortex did not show anysignificant variations in the number of cells whit any of the dies. We interpretedthis finding as a resistance to the cells to short periods of ischemia due tohypotension. The CA1 area of the hypocampus showed a gradual cellular lostrepresented as a decrease in the number of live cells (Nissl stain) at day 14 incomparison with day 1. This is correlated with the number of fluorojade positivecells that showed an important number of cells affected at day 1. In other words,the cells that were affected at day 1 (fluorojade C) are dead at day 14 for thisreason the number of live cells decreases at day 14 (Nissl stain). This mechanismcould explain why some patients, specially elderly patients show some cognitiveimpairment several days after surgery. The present observations suggest thatexposure to repeated hypotensive episodes lead to hippocampal damage. Patients withhemodynamic TIAs, cerebral arteriosclerotic disease, or orthostatic hypotension mayexperience repeated nonfatal circulatory deficiencies (Yap et al. 2008). Our results suggest that in this rat hemorrhagic model, briefperiods of hypotension result in neuronal damage or distress in the hippocampal CA1region one day after insult. By day 14, surviving cells are significantly reduced inthe hippocampus. We did not find any significant changes in cortical cells. Cellularlost is significant at day 14th and not at day 1 or 4th. We believe that apoptosisis an important factor that is responsible al least in part for this delay cellularlost.
                  Figure 4

                  Nissl-positive label in the (A) Cortex and (B) CA1 region, in controlgroup, 1, 4 and 14 days following hypotension. Nissl positivelabeled was statistically significantly higher in the CA1 region of rats 14day after hypotension as compared to control rats *p < 0.05.Nissl-positive label in the (A) Cortex and (B) CA1 region.Panel A shows the number of Nissl positive cells in frontal cortex.This is no difference in the number of cells at any of the time points. Thissuggests that the insult was not enough to cause cell dead. Panel Bshows the number of Nissl positive cells in the CA1 hypocampal area. Thenumber of Nissl positive cell decreased significantly 14 days after theinsult. This finding suggest that the cells where damage with the insult anddie progressively and this change becomes evident at day 14th.

                  In order to induce a hypotensive state, eight to twelve mL of blood were withdrawn toachieve a mean arterial blood pressure below 20 mmHg. The mean arterial bloodpressure (MAP) for all the groups was 19.2 ± 1.1. Yamauchi(Yamauchi et al. 1990;1991) foundhistopathological changes after one week in the hippocampus of rats with an averageMAP of 25 over a time frame of 2 or 3 min. Our data support the results shown byYamauchi and colleagues and show that hystological damage continues to be presenttwo week after the hypotensive insult.

                  The forty-eight point neurological scale did not show any statistically significantchanges in motor skills. Interestingly, we found temporal paralysis in the upperbody that lasted a few hours. At the time of the neurological evaluation, theparalysis had disappeared and could not be recorded as a positive finding. Althoughstatistically insignificant, we found that after 24 hours of recovery several ratsdemonstrated palpebral ptosis. By day 4, all rats that had palpebral ptosisrecovered. The dissection of the internal carotid artery typically manifests as anoculo-sympathetic palsy (myosis and palpebral ptosis) in humans (Eschmann et al.2006). We placed the catheters in the common carotidartery but since the surgical area is small, it is possible that we had manipulatedthe internal carotid artery leading to this finding.

                  The passive avoidance paradigm has been used for memory evaluation. Bekker et al.(2009) used this paradigm in nitroglycerine-inducedhypotensive and showed disruption in consolidation of long-term memory. In thepresent study we did not find any memory alterations. We hypothesize that thisdisparity is due to differences with the methods used to induce hypotension, as weused a hemorrhagic model to induce hypotension and Bekker and colleagues usednitroglycerine. Another difference between our studies is the time of hypotension.Bekker caused the hypotension early after the learning with the latest injection ofnitroglycerine given 3 hours after the training. Our learning regimen took place 24hours before hypotension and was tested twenty-four hours after hypotension. Thisstudy demonstrated that although there is damage in the hippocampus after 3 separateperiods of profound hypotension, memories that are already consolidated were notaffected.


                  Our conclusions suggest that accidental cerebral blood circulation impairment, whichmay happen for example during surgical procedure with general procedure with generalanesthesia in humans, needs to be considered carefully even in the absence ofclearly evidenced functional signs of neurological damage.


                  Authors’ Affiliations

                  Department of Molecular Pharmacology and Physiology, University of South Florida
                  Department of Neurosciences, University of South Florida
                  Tampa General Hospital
                  Department of Biomedical Sciences, Physiology Lab, University of Padova


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