Hippocampal cell loss in transient global cerebral ischemia in rats: a critical assessment

Hippocampal cell loss in global ischemia

Pergamon PII: S0306-4522(99)00163-3

Neuroscience Vol. 93, No. 1, pp. 71–80, 1999 71 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00

HIPPOCAMPAL CELL LOSS IN TRANSIENT GLOBAL CEREBRAL ISCHEMIA IN RATS: A CRITICAL ASSESSMENT ˜ O†‡ M. J. HERGUIDO,* F. CARCELLER,* J. M. RODA* and C. AVENDAN *Neurosurgery Service, “La Paz” University Hospital, Madrid, Spain †Department of Morphology, Medical School, Auto´noma University, Madrid, Spain

Abstract—The induction of transient global cerebral ischemia by permanent vertebral occlusion and temporary carotid ligation (four-vessel occlusion) is widely accepted as a valid tool for the study of pathogenesis and treatment of ischemia. The neural damage inflicted by this intervention is often assessed by measuring pyramidal cell loss in the CA1 hippocampal field. Nevertheless studies using this model in rats often fail to control variables that are relevant to the outcome, and/or apply biased methods to quantitate histological damage. We have applied unbiased stereological methods to estimate absolute numbers of surviving neurons in CA1 in Wistar rats subjected to either 10 or 20 min global ischemia using the Sugio et al. variant of the original four-vessel occlusion model. Animal mortality was high at both times, with neuron losses averaging 39% and 31%, respectively. Post-operative mortality was reduced substantially by using decompressive craniectomies and, even more effectively, by pre-treating the rats with low doses of phenytoin. Both maneuvers led to a severely increased CA1 neuron loss, which reached 50%, after an ischemia of 10 min. This finding strongly supports that mortality biases the sample. Other noteworthy findings that emerged from this study were a linear relationship between per-ischemic blood pressure increments and animal survival, and a negative correlation between cell survival and preferentially left-sided damage. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: CA1, cerebral ischemia model, hippocampus, phenytoin, stereology.

Experimental transient global cerebral ischemia in animals has been widely used to explore the pathogenesis and treatment of ischemia in humans through the development of different experimental procedures aimed at producing wellcontrolled cerebral ischemia in animals. A model which induces transient global ischemia in rats by occluding the four common carotid and vertebral arteries (the four-vessel occlusion [4-VO] model) has perhaps been the most widely used, since it was first described by Pulsinelli and Brierley. 39 Despite the unquestionable advantages of this model, it has two drawbacks that hamper its acceptance when an accurate measurement of post-ischemia brain damage is desired: the high mortality in spontaneously breathing animals, and the difficulties in histologically evaluating neuronal loss, even in so restricted a brain region as the hippocampus CA1 field. Mortality rates published in the original description of the global cerebral ischemia model were, respectively, 10% and 40% after 20 min and 30 min of bilateral carotid occlusion. 40 These rates were higher (up to 75%) in other laboratories using a similar model. 50,58 In many instances, however, information regarding rates, causes and timing of animal mortality is not given. Histological damage in ischemic tissues is evaluated by a variety of procedures. Many attempt to quantitatively estimate damage through different morphometric methods, in order to make valid statistical comparisons among groups of animals sustaining different ischemic insults or subjected to various neuroprotective therapies. However, by and large, these attempts have failed because they used biased quantitative methods. Classic morphometric methods cannot, in

general, elude biases due to the histological artifacts introduced by tissue processing, to failures in appropriately recognizing the target, and to systematic errors in the sampling and/ or counting design. Moreover, the wide variety of methods used make it difficult to compare reports from different authors. This study incorporates recently introduced stereological methods. They provide valuable counting and measuring tools which eliminate the need to introduce unreliable correction mechanisms, offer the possibility to ascertain the precision of the estimation, and enable accurate and unbiased total cell counts to be estimated with high efficiency. 18,19,64 The application of these methods has made it possible to analyse and eliminate factors that contribute to bias the estimations, thus helping to control and validate the 4-VO experimental model.

EXPERIMENTAL PROCEDURES

Animals and surgery Eighty-one young adult female Wistar rats (Granjas Jordi, St Vicens del Horts, Barcelona, Spain), weighing 265^85 g, were used for this study. They were divided into five groups: In Group 1 (nˆ6) rats were subjected to a simulated surgical procedure without cerebral ischemia and served as normal controls for histological analysis. In the remaining groups, transient global forebrain ischemia was induced for 20 min (Group 2, nˆ37), 10 min (Group 3, nˆ14), 10 min with the rats subjected to a decompressive posterior fossa craniectomy and vermiectomy (Group 4, nˆ11), and 10 min with the animals protected with low doses of phenytoin (Group 5, nˆ13). Animals were intraperitoneally anaesthetized with 2.5 ml/kg of a solution of diazepam (2 mg/ml), ketamine hydrochloride (Ketolar w, 25 mg/ml) and atropine (0.1 mg/ml). Anaesthesia was maintained, when necessary, with an additional one-third of the initial dose. Rats received no mechanical respiratory support at any moment of the procedure. A midline occipital-suboccipital incision was performed with separation of the paraspinal muscles. With the aid of the operating

‡To whom correspondence should be addressed. Abbreviations: CE, coefficient of error; CV, coefficient of variation; EEG, electroencephalogram; MABP, mean arterial blood pressure; SRS, systematic random sampling; 4-VO, four-vessel occlusion. 71

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microscope and bipolar electrocoagulation, vertebral arteries were exposed between C1 and C2, cauterized and subsequently severed, 55 in animals belonging to Groups 2, 3, 4 and 5. Twenty-four hours later, animals were subjected to ether inhalation through a mask placed over the muzzle and allowed to breathe spontaneously. Both common carotid arteries were dissected in the neck through a longitudinal midline incision. After a tracheostomy to ensure airway integrity during the whole procedure, both common carotid arteries were occluded by ligatures, again in Groups 2, 3, 4 and 5. At this point, the masks were removed, suppressing ether inhalation, and the animals began to breathe room air spontaneously. After bilateral carotid occlusion had been maintained for either 10 or 20 min, depending on the group, recirculation was restored by release of the carotid ligatures. In all cases blood flow was confirmed under the operating microscope. During the entire ischemia-reperfusion procedure, mean arterial blood pressure (MABP) and pulse rate were monitored through a heparin-filled polyethylene catheter inserted in the right femoral artery. Arterial blood gases and plasma glucose concentrations were measured before carotid occlusion, during ischemia, and 5 min after reperfusion. Thermostatic probes, coupled to a servocontrolled heating plate and lamp, were placed in the temporalis muscle and rectum to ensure that cerebral and rectal temperatures remained at 36.5^0.58C and 37.0^0.58C, respectively. A bipolar electroencephalogram (EEG) was recorded during ischemia and 10 min after reperfusion by one reference central scalp needle electrode and two active lateral electrodes. Animals belonging to Group 5 were treated with low doses of phenytoin. The drug was subcutaneously administered twice, at a dose of 5 mg/kg each time, immediately after the first surgical procedure of vertebral artery electrocoagulation and sectioning, and then after release of the carotid ligatures 24 h later. Plasmatic concentrations peaked between 30 and 60 min after administration, and did not exceed 7 mg/ml in any case. Rats were then placed in an incubator at 268C with free access to water and food, and were closely monitored during the three-day survival period for seizure activity, neurological status, and cardiorespiratory arrest and secondary death. Animals with no access to water due to neurological impairment were daily injected with 10 ml of saline solution subcutaneously in order to avoid dehydration. Histological analysis After the survival period was completed, animals received an overdose of pentobarbital, and were perfused through the heart with 200 ml of isotonic saline solution, followed by 600 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, using a peristaltic pump at a mean rate of 40 ml/min. The brains were removed from the skull and kept refrigerated in the same fixative for at least one week. Subsequently, they were progressively dehydrated in ascending concentrations of ethanol solutions, and embedded in low-viscosity nitrocellulose (celloidin, Fluka). The blocks containing the brains were serially sectioned parallel to the coronal plane on a sliding microtome with the thickness set at 40 mm. The real mean thickness of the sections during cutting was estimated by separately sectioning a small block of celloidin of a known height. Additionally, optical depth measurements of the thickness were performed on the mounted and coverslipped sections using a microcator. Every fifth section was stained with 0.5% Cresyl Violet, serially mounted on glass slides and coverslipped using DePeX mounting medium. Mortality and exclusion criteria Mortality rates in each group were classified as: “peri-operative”, referring to the animals that died during carotid surgery (no animals died during vertebral occlusion), or within the first hour post-surgery (“during” and “immediately after” the operation, respectively), and “post-operative” mortality, all animals dying after that. The possible factors related to mortality were evaluated (see below). Exclusion criteria, which were established a priori and strictly adhered to, were as follows: (i) those animals which required persistent assisted pulmonary ventilation during the peri-operative period for more than 2 min immediately after occlusion (nˆ5, nˆ2, nˆ1, nˆ2, for Groups 2, 3, 4 and 5, respectively), (ii) those whose EEG did not show an isoelectric pattern after bilateral carotid occlusion (nˆ0 for all groups), and (iii) those developing seizures during the post-operative period (nˆ7, nˆ0, nˆ0, nˆ0, for Groups 2, 3, 4 and 5, respectively). All other animals were included in the study.

CA1 delineation The CA1 field of the hippocampus was delineated according to wellestablished criteria. 4,5,64 CA1 is readily identifiable in Nissl-stained sections (Fig. 1). Its lateral border with CA2 is recognized by the thickening of the pyramidal cell layer in CA2, and it is sharp along the whole dorsal (or septal) half of the hippocampus. Although CA2 is harder to distinguish from CA3 on coronal sections in the rest of the hippocampus, the beginning of CA1 is distinguished by the more compact arrangement of mostly smaller cell bodies, particularly at the upper surface (stratum radiatum) of the pyramidal cell layer. On its other end, the transition with the subiculum is less distinct, and is more difficult to recognize since the coronal sections are tangential to the caudal CA1. Still, the CA1 pyramidal cell layer is marked by the presence of tightly packed pyramidal cell bodies in a layer four to five cells deep. Toward the subiculum, the uppermost cells of the layer become progressively more loosely packed, and the border is defined as the point at which the cells of the pyramidal cell layer cease to form a continuous row. 64 This border more or less coincides with the point where the upper convex curve of the CA1 pyramidal cell layer changes into the upper concave curve which characterizes the subiculum. The stratum radiatum also helps to distinguish CA1, since it is deeper and more sharply separated from the pyramidal cell layer in CA1 and the stratum lacunosum-moleculare than in the adjacent subiculum and CA2/CA3 fields. Cell count The whole sampling and counting procedure in this study was carried out with the help of an interactive computer system consisting of a high-precision motorized microscope stage, including a planachromatic ×2.5 (o ×4) dry lens (Olympus, SPlan ×2.5) and a planapochromatic ×100 oil-immersion lens with n.a.ˆ1.40 (Olympus, SPlan Apo100), a 0.5 mm resolution microcator (Heidenhain VZR 401), a solid-state video camera and a high resolution video monitor. The interactive test grids and the control of the motorized stage were provided by the GRIDw General Stereological Software Package (Olympus, Denmark) running on an Amiga 2000 computer. The total number of surviving neurons in the pyramidal layer of CA1 was obtained using an unbiased stereological method, the “optical fractionator”, which combines the “optical disector” 19,54 with a “fractionator” sampling scheme. 62,63 Although most of the neurons in this layer correspond to pyramidal cells, the count includes a small population of interneurons whose somata were located within this layer. The measuring method has been described in detail elsewhere. 2,63,64 Essentially, the procedure consisted of counting the number of nucleoli within morphologically normal pyramidal cells contained in a series of systematically random sampling volumes, known as optical disectors, through the CA1 in both hippocampi. In practice, this consists of counting the number of nucleoli that come into view as one focuses through a known distance in the thickness of the section at the sampled locations. The figures obtained (SQ 2), are divided by the fraction of the total volume of the CA1 subfield represented by the sum of the sampled volumes (fT). This final or total sampling fraction is the product of the fractions at each of the three steps of the sampling procedure, fT ˆ fs :fd :fh were fs is the numerical fraction of sections used (1/10, corresponding in most cases to nine to 10 sections separated by identical intervals, with the first one picked at random from those falling within the first interval width); fd is the areal fraction of the target region covered by all the sampling frames or disectors (1/40, with the counting frames having been distributed as well in a systematically random fashion through the CA1 region in each section); and fh is the linear fraction of the section thickness covered by the height of disector frame (1/2.5 on average, corresponding to a disector height of 15 mm and a mean section thickness of 38 mm). The actual section thickness was assessed at various points of each section by focusing through the section with a ×100 oil-immersion objective, and reading the distance given by the microcator between the upper and bottom surfaces. The mean values were about 5% less than the nominal 40 mm of the microtome setting. The resulting absolute number of neurons (N) is, then, the product of the nucleoli counts made in all the disectors (SQ 2) and the reciprocal of the total sampling fraction (fT): N ˆ SQ2 ·1=fT

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Fig. 1. Cresyl Violet-stained, celloidin-embedded coronal brain sections at the rostral (or dorsoseptal: A), intermediate (B) and caudal (C) levels of the hippocampus, obtained from a sham-operated animal. CA1 is continuous medially with the subiculum and laterally with the small CA2 sector (arrows in A and B). Its border with CA2 is recognizable by a sharp thickening of the pyramidal cell layer in the latter sector. The transition with the subiculum can be identified by the loosening of the tightly packed pyramidal cell bodies, which cease to form a continuous row. At caudal sectors (C), only the caudal shell of CA1 appears, and it is then bounded dorsally and ventrally by the subiculum.

The precision of the estimates of N obtained for each brain was evaluated by computing the coefficient of error (CE) for the counts (SQ 2); this is a function of two independent factors, the variance of the nucleoli count for each section (the so-called “Nugget effect”), and the variance of the counts across all systematically random sampled sections (SRS). 20,62 The general formula for the CE thus defined is:

CE…

X

q ÿ
Nug 1 VarSRS 2 P 2 Q †ˆ Q

The sampling scheme used was designed to keep the value of CE(Q 2) at or below 10%.

Data analysis The physiological and metabolic parameters, measured before, during and after arterial occlusion, were compared using an ANOVA test. Cell counts in CA1 for each case and hemisphere were expressed as absolute mean^S.D. values. Results for each ischemic group were systematically compared with the sham-operated group using an ANOVA test, followed by post hoc Dunnett’s t-test. The correlation between the number of surviving hippocampal cells and the left/right ratio was established using Spearman’s correlation coefficient. Values corresponding to the EEG register were compared using an ANOVA test. Significance was assumed at P,0.05 for all the estimations. The relation between MABP increments and eventual mortality was analysed by calculating the area under the Receiver Operating Curve (ROC), 21 and was predictive with a 95% confidence level. Sensibilities and specificities were obtained for different cut points in MABP increment. Those cases in which the interval did not include the value 0.5 were considered statistically significant.

RESULTS

Clinical and metabolic status Physiological parameters monitored immediately before ischemia (MABP, heart rate, blood gases and plasma glucose concentrations) showed no statistical difference between groups (Table 1). All rats suffered a weight loss of aproximately 10% in the post-ischemic period. Post-operative epileptic seizures were monitored during the three-day survival period, or until animal death. They were observed in seven (19%) of the animals subjected to ischemia for 20 min, only two of which (5%) survived. No seizures were observed in Groups 3, 4 and 5. Mortality No rat of Group 1 died before perfusion. Peri- and postoperative mortality were, respectively, 34% and 50% in Group 2, 29% and 29% in Group 3, 25% and 17% in Group 4, and 31% and 0% in Group 5 (Fig. 2). Correlation between acute blood pressure response and animal survival We have analyzed the MABP increment as a predictive factor of global mortality. This increment was calculated according to the formula: %I ˆ ‰…MABP2 2 MABP1 †=MABP1 Š × 100

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M. J. Herguido et al. Table 1. Physiological parameters before, during and after carotid occlusion for Groups 2–5 Parameters Mean arterial blood pressure (mmHg) Group 2 Group 3 Group 4 Group 5 Heart rate (BPM) Group 2 Group 3 Group 4 Group 5 Arterial pO2 (mmHg) Group 2 Group 3 Group 4 Group 5 Arterial pCO2 (mmHg) Group 2 Group 3 Group 4 Group 5 Arterial pH Group 2 Group 3 Group 4 Group 5 Hemoglobin (g/dl) Group 2 Group 3 Group 4 Group 5 Arterial blood glucose (mg/dl) Group 2 Group 3 Group 4 Group 5

Before

During

After

91.43^13.52 88.08^16.34 94.40^13.23 86.31^9.54

134.83^29.46 159.20^16.56 148.30^27.28 146.20^29.30

113.35^16.12 130.50^13.76 107.22^13.06 104.80^34.16

376.67^47.07 351.69^106.03 415.00^37.75 391.50^33.50

402.73^42.92 379.09^133.39 400.00^8.66 450.00^17.32

439.09^52.00 412.80^116.44 345.00^21.21 410.63^126.50

116.89^16.52 103.49^20.60 109.15^17.50 112.62^27.99

115.97^21.93 124.17^16.39 112.44^22.43 127.70^21.72

106.80^19.82 108.54^20.53 104.09^12.04 110.33^13.54

25.01^5.80 26.75^7.01 23.29^3.19 27.31^6.51

21.66^7.92 17.93^6.77 20.84^8.10 17.53^6.97

21.70^8.51 21.54^4.84 22.21^4.90 20.84^5.20

7.32^0.06 7.33^0.08 7.36^0.09 7.34^0.07

7.37^0.08 7.37^0.11 7.38^0.06 7.38^0.06

7.36^0.08 7.33^0.10 7.37^0.03 7.37^0.06

11.48^3.63 11.52^2.17 12.10^1,54 11.05^2.33

11.24^1.52 11.00^1.77 11.83^1.60 10.38^1.47

10.44^1.30 10.69^2.71 14.70^7.03 11.46^2.71

149.54^66.04 143.69^51.98 132.18^49.06 138.46^55.25

165.75^71.53 128.91^57.29 150.11^37.95 123.00^41.81

156.74^77.84 142.82^49.16 155.88^52.70 138.11^17.70

Values are presented as the mean^S.D. Parameters were measured 10 min before occlusion (basal values), 5 or 10 min after occlusion for groups subjected to 10 or 20 min of ischemia, respectively, and 10 min after reperfusion. Basal values did not show statistical differences between groups.

and 149.2^27.1 mmHg, respectively (Mean^S.D.), reflecting a mean increment, %Iˆ67^33%. We found a positive correlation between animal survival and increments in MABP within the 60–120% range, when all groups were considered together. However, the highest MABP increments (over 120%) showed a tendency to be associated with higher mortality rates, although not significantly, probably because too few animals presented these levels to be significant. 26 When MABP was evaluated during the whole procedure (before, during and after ischemia) a linear increment was found. This increment fits the formula: MABPi ˆ 109 1 …7:88 × timei † Fig. 2. Animal mortality differed markedly between groups. Survival (hatched bars) was maximal in the sham-operated animals, but dropped to less than 20% in animals subjected to 20 min ischemia (Group 2). Peri-operatory mortality (empty bars) was roughly the same in all ischemic groups. Post-operatory mortality (solid bars) decreased from 50% in Group 2 to zero in animals pretreated with phenytoin (Group 5).

where MABP1 represents the basal value of arterial pressure before occlusion and MABP2 represents the same parameter measured 1 min after occlusion. The values were obtained from all the animals together and were 90.8^14.5 mmHg

This formula is only valid for the 0–5 min interval after carotid occlusion. By substituting the term “timei” for the corresponding value, the expected MABP may be empirically predicted. Neurophysiology All animals displayed an isoelectric EEG during and immediately after ischemia. Ten minutes after starting reperfusion, only a fraction of the animals (22%, 50%, 25% and 0% in Groups 2, 3, 4 and 5, respectively) had recovered a normal EEG pattern. No correlation was found, however, between

Hippocampal cell loss in global ischemia

75

Fig. 3. Cresyl Violet-stained coronal sections obtained from animals belonging to different ischemic groups and showing various patterns and degrees of hippocampal damage. Arrows point to the CA1–CA2 border. (A) Severe neuronal damage restricted to CA1 in a rat subjected to 10 min global brain ischemia (Group 3). (B) Apparently intact dorsal hippocampus, including CA1, in a rat subjected to 20 min ischemia (Group 2). (C) Severe neuronal damage affecting all CA fields in a dorsocaudal sector of the hippocampus from an animal belonging to Group 4.

EEG recovery and animal survival or total number of surviving neurons. Histopathology In all animals with substantial neuronal loss, the lesion was predominantly located in the dorsoseptal region of CA1, with the most ventrotemporal areas showing a more normal pattern and cellular integrity. Overt histological damage, however, adopted variable patterns, from the frequent finding of a continuous band of complete neuronal loss stretching mediolaterally between the subiculum onto CA3, to the less common presence of interspersed patches of damage within an otherwise apparently intact CA1 pyramidal cell layer. Also, the caudotemporal spread varied extensively (Fig. 3). The histological appearance of the ischemic hippocampus was characterized by the presence of “ghost” and “shrunken” neurons often alternating with other cells that were considered viable because of the presence of at least one nucleolus within a clear nucleus displaying an apparently intact nuclear membrane (Fig. 4). Neuron counts From 50 to 400 apparently viable neurons (SQ 2) were counted in each hippocampus, depending on the degree of damage caused by the ischemia. The total mean number of pyramidal neurons in CA1 in

controls (Group 1) was 763,000, with a low inter-animal variance (coefficient of variation [CV]ˆ0.06; Table 2). This figure closely matched the one reported in a previous report that applied similar stereological methods in the same species and strain 64 (but see Discussion). In the ischemic groups, the mean values ranged between 388,000 and 524,000, with much larger inter-animal variances (CVs between 0.32 and 0.59). Differences were significant between Group 1 and each of the ischemic groups (Table 2). In spite of the fact that no significant differences could be found between the ischemic groups, Groups 2 and 3 tended to exhibit less hippocampal cell loss than Groups 4 and 5, though longer periods of ischemia were employed in the former. This statistical tendency to exhibit less severe damage appears precisely in the groups with higher death rates, suggesting that survival in Groups 2 and 3 is somehow paralleled by a relatively better resistance to global brain ischemia. This strongly suggests that high mortality biases the sample, and that the sample cannot then be considered representative of the general population. Differences between hemispheres were negligible in controls, and in ischemic rats with total cell counts over 600,000 (left/right differences were, on average, less than 3% in each case). In ischemic animals sustaining higher neuron losses, however, there was a statistically significant negative correlation between the absolute numbers of surviving cells and a preferential cell loss in the left hippocampus, with this side having about 16% fewer neurons, on average,

76

M. J. Herguido et al. Table 2. Absolute counts of surviving neurons in field CA1 of the hippocampus Left

Group 1 (nˆ6) Group 2 (nˆ5) Group 3 (nˆ6) Group 4 (nˆ6) Group 5 (nˆ8)

M^S.D. 376^25 215^89 260^89 221^141 178^116

Right CV 0.07 0.42 0.34 0.64 0.65

M^S.D. 387^28 253^108 264^79 229^125 210^121

Both CV 0.07 0.43 0.30 0.55 0.58

M^S.D. 763^45 468^196 524^166 450^264 388^198

CV 0.06 0.42 0.32 0.59 0.52

Values are presented as the mean^S.D. (×10 23). Significant differences were found between Group 1 and all the ischemic groups (ANOVA test followed by post hoc Dunnett’s t-test, P,0.05).

Characteristics of the experimental model

Fig. 4. High-power photomicrographs of ischemic CA1 showing characteristic histopathological images after a three-day survival period. (A) Pyknotic, shrunken and “ghost” profiles, with a few, interspersed, apparently intact pyramidal cells (arrows). (B) More severely damaged area where only one surviving neuron (apparently non-pyramidal) can be identified. Scale barˆ40 mm.

than the right side. At single case level, these differences ceased to exist (they were less than 5%) when the number of surviving cells was under 200,000.

DISCUSSION

The 4-VO rat model of transient global ischemia, introduced by Pulsinelli and Brierley in 1979, 39 soon became very popular because of its advantages over alternative procedures. Since it does not need hemovolemic- or pharmacologically-induced hypotension to produce brain damage, the 4-VO model avoids involvement of organs other than the brain in the ischemic process. Diverse methods have been used to evaluate the tissue damage inflicted by the ischemia and/or reperfusion. 10,17,33,42,49,58 The most widely used method, however, is the quantitative evaluation of the histological damage, which is often regarded as a direct and objective measure of the ischemic tissue injury. Pyramidal cell loss in CA1 has been repeatedly described and quantified ever since the first models of transient global ischemia were introduced. However, the existing plethora of global ischemia protocols and quantitative methods hamper their comparison or synthesis, mostly because of actual differences in the models themselves or insufficient controlling and/or reporting of significant variables, and of methodological shortcomings in the quantitative analysis (cf. Herguido, 1999). 25

Our study employs the modification introduced by Sugio et al. 55 to avoid the risk of defective vertebral artery occlusion which is not uncommonly associated to a blind surgical approach. There has been a tendency to reduce the duration of the ischemic period from 20 min 39 to 10 min or even less. In the present study we analysed the increase in animal morbidity and mortality that accompanied longer periods of ischemia, and also found that high death rates bias the results (Fig. 2, see below). Post-ischemia survival periods are, most often, three or seven days. The histological lesion in CA1 is first visible with light microscopy 24 h post-ischemia; damage peaks at 72 h, and little, if any, increments seem to appear between 72 h and seven days. 14,42,51,61 Appropriate control of a number of physiological parameters that are known to affect the ischemic damage is mandatory, in order to ensure a correct application and good replicability of the model. Nevertheless, many reports fail to control and/or report these parameters. 25 Brain temperature during the ischemia is an important factor that can condition the extent of neuronal lesion in experimental models. 13,15 Rectal and cerebral temperatures must, therefore, be kept within a narrow range, so that the uncontrolled and potentially significant effects of temperature change do not affect the outcome. An isoelectric EEG reading during carotid occlusion is considered a reliable index of severe interruption of cerebral blood flow in anaesthetized animals. Plasma glucose levels should also be controlled, because of the well-known fact that the brain glucose concentration at the moment of cerebral hypoxia-ischemia determines the severity of the resulting ischemic brain damage. Animals made hyperglycemic before severe or during moderate brain ischemia, present greater neurological deficits, 44,53 morphologically more severe brain damage, 30,43,44 and a poorer recovery of their glucose metabolism 16,52 and high energy metabolites, 46,60 than do normoglycemic controls. The well-known hypertensive reaction to ischemia is also quite relevant: our data showed that a MABP increment of between 60% and 120% after bilateral carotid occlusion was probably, among all the other physiological variables, the best predictor of animal survival. 26,27 Quantitative histological assessment of brain damage in CA1 Although a majority of histological studies focus only on the dorsal hippocampus, or even on restricted sectors of this region, there are reasons to include the whole CA1 field in the study. There is a gradient in vulnerability to ischemia between the septal and temporal ends of the hippocampus, but a

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Table 3. Comparative analysis of the results obtained by different authors employing similar forebrain ischemia models and histological evaluation procedures Mean number of viable neurons/mm Author(ischemic/ survival period) Benveniste et al. 3 (10 min/four days) Lin et al. 34 (10 min/three days) Wengenack et al. 61 (12 min/three days)

Sham animals

Ischemic animals

180 (0.07)*

33 (1.8)*

162 (0.07)

42 (1.57)

234 (0.03)*

6 (1.38)*

Author(ischemic/ survival period) Haseldonckx et al. 22 (9 min/seven days) Johansen and Diemer 29 (10 min/eight days)

Sham animals

Ischemic animals

–

55.5 (?)

115 (?)*

31 (?)*

Percentage of necrosis (%)† Author(ischemic/ survival period) Buchan et al. 8 (5 min/seven days) Ashton et al. 1 (8 min/seven days) Johansen and Diemer 29 (10 min/eight days) Henrich-Noack et al. 24 (10 min/seven days) Buchan et al. 9 (10 min/seven days)

Results reported

Author(ischemic/ survival period)

Results reported

43 (0.7)

Rod and Auer 48 (10.5 min/seven days) Ordy et al. 38 (15 min/six days) Buchan et al. 8 (15 min/seven days) Heurteaux et al. 28 (20 min/seven days)

91 (0.13)

52 (0.51) 73 (?) 76 (0.09)

82.5 (?) 92.5 (0.04) 78 (?)

82 (0.15)

Results are expressed as mean (CV). *Estimated from original report. †Most of these studies focus the target area on the “dorsal” hippocampus. In the rest this information is not specified.

defined boundary between the dorsal and ventral areas does not exist. Therefore, cropping only a part of the hippocampus would result, at best, in a loss of precision, and, at worst, in a clear cut bias. As to the sampling method for the sections within the target region, the frequent use of a few sections at preselected levels jeopardizes the unbiasedness of the method. Independent random sampling throughout the whole field is in principle bias-free, although much less efficient than systematic sampling. 20 The selection of counting units is not a minor issue, since in practice there is always a degree of uncertainty regarding the pre-established viability criteria. Choosing to count ischemic neurons surely leads to an under-counting bias, because some of the neurons will have disappeared, or have become “ghost” cells, which are difficult to identify and distinguish from fixation artifacts. This is already obvious at 72 h after the ischemic event, but even more so at longer survival periods. Moreover, except for one study that reports absolute numbers of surviving cells, 37 most studies report cell densities in various ways, as a number of ischemic, or of surviving neurons per unit length or area of pyramidal cell layer, or as a percentage of ischemic neurons, etc. (Table 3). Alternatively, results have been expressed by using grading to score cell damage; 11,12,38–40,57,59,61,65 such an approach may be excessively weighted by subjectivity. Another approach that can seriously affect result validity is estimating numerical neuron density employing one- or twodimensional probes to explore a volume. 54 By itself this approach causes bias, because the probability that a neuron or its nucleus appears in a section depends on its size, shape and spatial orientation. Moreover, artifacts such as split nuclei, “lost caps”, overprojection phenomena and tissue shrinkage, also contribute to bias, 2,63 particularly in the relatively thin sections (less than 20 mm) used in almost every

paper reviewed. The use of classical correction factors to partially avoid over- or under-counting is not a common practice.

Intra- and inter-individual variability The total number of neurons in a particular subdivision of the hippocampus of a normal or ischemic animal, is subject to ordinary biological or inter-individual variation. However, if estimates, and not absolute values are used, as occurs in most neurobiological cell counts, then the final observed variance will include a component of error in the estimate for each case. This internal variance is produced by the overall sampling scheme, and depends on the number of sections per animal and fields of vision in each section, as well as the number of neurons counted in each field of vision. It represents the precision of the estimates made in any particular case, and can be expressed as the CE of the estimate. In order to ensure a minimum of efficiency in the procedure, the contribution of the precision error (as expressed by the squared CE) to the final observed variance should be kept below 50%; if this is done, the latter will confidently reflect the true inter-individual or biological. 2,63,64 In the present report the authors have ensured a minimal contribution of internal variance to the final variance by maintaining a CE below 10%. The CE is not mentioned in any of the papers we have seen. In addition to this methodological “noise”, true intraindividual variations are found and may depend on side-toside and rostrocaudal differences in the surviving neurons. Both differences have been reported often, and here we show that the preferential vulnerability to ischemia of the left hippocampus is only true within certain degree of

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ischemic injury, since both sides are equally affected in animals with very severe damage. Inter-individual variability in our study is the lowest reported for intact and sham-operated animals. Our cell counts for a single hippocampus (mean left/right absolute neuron count^S.D.ˆ381,500^26,500; CVˆ0.07; nˆ6) match those obtained in normal animals by West et al. 64 using stereological methods (mean unilateral absolute neuron count^S.D.ˆ382,000^73,000; CVˆ0.19; nˆ5), and by Boss et al. 5 using cell density estimates and Abercrombie’s correction for split nuclei (mean unilateral absolute neuron count^S.D.ˆ320,000^36,000; CVˆ0.11; nˆ4). On the other hand, the only other study with Wistar rats that provided absolute cell counts in CA1, using reliable stereological methods, 37 also found low inter-individual variability (CVˆ0.11), but reported about 25% fewer neurons in their “sham” group. This seems difficult to explain, but two factors may have played a role in the discrepancy: 10 of the 28 “sham” animals used by Olsen et al. 37 had undergone bilateral vertebral cauterization and were allowed to survive at least 10 days before sacrifice; if this manipulation impaired hippocampal perfusion to any extent, the resultant cell loss, however slight, would have lowered the final mean value. Also, it is possible that these authors were too conservative in setting the CA1-subiculum boundary, and thus excluded some CA1 cells from their count. In contrast with control animals, variations among cell counts are very high in all the ischemic groups, as reflected by CVs ranging between 0.32 and 0.59 (Table 2). Other studies find much higher variability among ischemic animals when linear cell counts are reported, but not when damage is presented as a percentage of necrosis (Table 3). Interindividual variability in intracranial arterial collateral circulation may be partly responsible for the observed variance in cell survival. Moreover, it could also explain the seemingly contradictory results obtained in the 20 min ischemic group (Group 2), in which the low percentage of surviving animals had lost fewer cells than the animals subjected to shorter ischemic periods in other groups. For all the above reasons, the histological results obtained by different authors employing more or less comparable models of forebrain ischemia are absolutely unequal. Such marked differences can not be explained by any reason other than bias in the sampling and quantification method (Table 3). Animal mortality and CA1 pyramidal cell survival Generally speaking, our mortality rates are higher, and surpass the figures published in the original description of the model, 39 where they ranged from 10 to 40%, for 20 and 30 min of bilateral carotid occlusion, respectively. This discrepancy may be attributed to two main factors: (i) the maintenance of normothermia during and after the procedure, and (ii) the certainty of vertebral blood flow interruption provided by Sugio et al.’s 55 modification to the classic 4VO model. 39,41,42 Both aspects have been discussed earlier in this paper. Of special importance to the model is the fact that animal mortality is a prominent cause of bias in quantitative evaluation of neuronal loss. We found that animals subjected to the most prolonged periods of ischemia (Group 2) showed the highest mortality rate (84%), as was expected. However, and rather surprisingly, the survivors in this group

tended to exhibit less hippocampal neuronal loss (mean cell countˆ486^196×10 3) than groups subjected to much shorter ischemic periods. Groups 4 and 5, subjected to 10 min ischemia, had cell counts ranging between 388^198×10 3 and 450^264×10 3, and showed the lowest mortality rates, 31% and 42%, respectively. This positive correlation between animal mortality and absolute cell counts indicates that, whatever their anatomical or biochemical advantages, surviving animals in the groups with the higher mortality rates belong to a particularly ischemia-resistant subpopulation. Animal mortality, then, must be reduced to a minimun, not only for ethical and practical reasons, but also in order to eliminate this source of bias. In principle, mortality can be reduced by keeping the operated animals under tight clinical and pharmacological control throughout the survival period. Nevertheless, we tried to decrease such control to a minimum for the sake of consistency and reproducibility, and the experiments were carried out on spontaneously breathing animals without surgical or pharmacological manipulations. Moreover, we were able to distinguish different circumstances responsible for animal death at distinct survival intervals, and this led to our criteria for “during”, “immediately after” and “delayed post-operative” animal mortality. Perioperative mortality is attributable to accidental conditions, such as airway obstruction from accumulated secretions, metabolic disorders, an absent or excessive MABP response to bilateral carotid occlusion and/or failure by vital structures in the brainstem, such as the respiratory and vasomotor centers. These early events seem independent of any structural or functional changes secondary to the chain of intracellular events triggered by ischemia and/or reperfusion, these probably being insufficiently developed at so short an interval as to provoke animal death. Delayed mortality, on the other hand, is more likely to be related to events directly connected to actual ischemia-reperfusion damage, and efforts should be mainly aimed at controlling this type of mortality without interfering in hippocampal cell loss. As shown in the Results, we tested two different procedures with good results: surgical decompression by means of occipital bone and cerebellar vermis removal, and preventive anticonvulsant phenytoin treatment. Although, as expected, the mortality rate during or immediately after surgery was similar in the four groups of ischemic animals, the incidence of delayed post-operative mortality was markedly decreased by these approaches. The idea of surgical decompression was based on the autopsy finding of generalized brain edema encountered in ischemic rats that died before the preestablished 72-h survival period, probably because of brainstem compression. Although not abolished, delayed mortality in this group of rats decreased to almost one-half that occurring in animals with the same time of ischemia (10 min) but lacking any protective maneuver. In an attempt to avoid the surgical aggression involved in a decompressive craniectomy, we targeted subclinical seizures as another possible cause of delayed mortality. Overt seizures during the post-operative period in our study were associated with prolonged ischemic periods and higher mortality rates (Group 2). Post-operative mortality was abolished in the group of rats treated with phenytoin. The antiepileptic effect of phenytoin was first identified 60 years ago 35 and the drug has been used extensively for convulsive disorders since. 47 In the last few years, some studies have shown that phenytoin also has a neuroprotective effect, although most of these

Hippocampal cell loss in global ischemia

studies used doses at least twice as large as those employed in the present study. 6,7,23,31,32,36,45,56 We think it unlikely that phenytoin protected the brain against ischemia/reperfusion damage in our study, not only because of the low dose employed, but also because the histological damage in the group of rats treated with phenytoin was even more severe. This suggests that the dose of phenytoin probably just avoids any subclinical epileptic activity (no overt seizures were noted in the groups of animals subjected to 10 min of ischemia), which, if left unchecked, would have reduced the chances of the animal surviving the ischemic insult. CONCLUSIONS

This study shows that the reliability and validity of an

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experimental global brain ischemia model is based on a strict control of the physiological, neurophysiological, and surgical characteristics of the experiment. Furthermore, the quantitative histological evaluation of the outcome greatly benefits from applying efficient and unbiased stereological methods. In addition to providing a reliable and reproducible measure of the insult, these methods enabled us to identify a source of bias in the estimation of ischemic damage.

Acknowledgements—The authors wish to thank Drs A. Carcas and B. Tabare´s for the pharmacological studies of phenytoin, Dr R. Madero for statistical analysis, and Ms C. F. Warren for style correction. This work was supported by FIS Grant 1177/96.

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