Pattern of neuronal loss in the rat hippocampus following experimental cardiac arrest-induced ischemia

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Abstract

The pattern of neuronal loss in the rat hippocampus following 10-min-long cardiac arrest-induced global ischemia was analyzed using the unbiased, disector morphometric technique and hierarchical sampling. On the third day after ischemia, the pyramidal layer of sector CA1 demonstrated significant (27%) neuronal loss (P<0.05). At this time, no neuronal loss was observed in other cornu Ammonis sectors or the granular layer of the dentate gyrus. On the 14th postischemic day, further neuronal loss in the sector CA1 pyramidal layer was noticed. At this time, this sector contained 31% fewer pyramidal neurons than on the third day (P<0.05) and 58% fewer than in the control group (P<0.01). On the 14th day, neuronal loss in other hippocampal subdivisions also was observed. The pyramidal layer of sector CA3 contained 36% fewer neurons than in the control group (P<0.05), whereas the granular layer of the dentate gyrus contained 40% fewer (P<0.05). The total number of pyramidal neurons in sector CA2 remained unchanged. After the 14th day, no significant alterations in the total number of neurons were observed in any subdivision of the hippocampus until the 12th month of observation. Unbiased morphometric analysis emphasizes the exceptional susceptibility of sector CA1 pyramidal neurons to hypoxia/ischemia but also demonstrates significant neuronal loss in sector CA3 and the dentate granular layer, previously considered ‘relatively resistant’. The different timing of neuronal dropout in sectors CA1 and CA3 and the dentate gyrus may implicate the existence of region-related properties, which determine earlier or later reactions to ischemia. However, the hippocampus has a unique, unidirectional system of intrinsic connections, whereby the majority of dentate granular neuron projections target the sector CA3 pyramidal neurons, which in turn project mostly to sector CA1. As a result, the early neuronal dropout in sector CA1 may result in retrograde transynaptic degeneration of neurons in other areas. The lack of neuronal loss in sector CA2 can be explained by the resistance of this sector to ischemia/hypoxia and the fact that this sector is not included in the major chain of intrahippocampal connections and hence is not affected by retrograde changes.

Introduction

Damage to the central nervous system is a decisive factor for the clinical outcome of a patient who survives clinical death. Sector CA1 of the cornu Ammonis (CA); layers 3, 5, and 6 of the neocortex; the striatum, and some thalamic nuclei are known to be areas of the brain that are selectively vulnerable to ischemic insult [1], [2], [3], [4], [5]. Evidence gathered from animal studies [1], [6], [7] and observations made of human material [8] indicate that neurons in these vulnerable areas die not during a brief period of ischemia but within a few days after reperfusion. According to current concepts, this phenomenon of delayed neuronal death is linked to a perturbation of calcium homeostasis leading to gradual postischemic rise in free cytosolic calcium concentration [9], [10], [11], [12], [13], which eventually produces damage to mitochondria̶mitochondria permeability transition [14], [15]. Free radicals released by endothelial cells, activated microglia cells, and even by damaged mitochondria are complementary injurious factors contributing to neuronal death [16]. Degeneration of selectively vulnerable neurons after global ischemia is morphologically nonapoptotic [17], although some evidence of apoptosis, such as DNA fragmentation, has been found in vulnerable brain areas [7], [18], [19], [20].

The hippocampus is the central structure of the memory system. Because of its unique vulnerability to ischemic insult and its simple anatomy, this structure is the model used most frequently to study ischemia-related phenomena. However, particular hippocampal subdivisions show different susceptibilities to ischemia/hypoxia. Sector CA1 is known to be the most sensitive area, and exposure to even short ischemic insult triggers mechanisms of delayed neuronal death, which results in marked neuronal loss in this structure [21]. Sectors CA2–3 were reported to be relatively resistant to ischemia. Prolonged ischemia results in reversal of ischemic changes in sector CA2–3 neurons, but apparent neuronal loss in these sectors has seldom been demonstrated [22]. The granular layer of the dentate gyrus appears to be the hippocampal subdivision that is the most resistant to ischemic insult. After 20–30 min ischemia, only transient neuronal swelling, not ischemic neuronal changes or neuronal loss, was noticed [22], [23]. However, most previous studies were based on qualitative features; hence, they were able to detect neuronal loss when it was significantly profound, but they were not able to assess absolutely its magnitude, timing, and rate of neuronal death.

In the present study, we analyzed the temporal pattern and magnitude of neuronal loss in various hippocampal subdivisions after 10-min-long cardiac arrest-induced ischemia. To assess neuronal loss, unbiased morphometric techniques were used. Because relative measures such as numerical density of neurons might not depict actual changes in number of neurons in atrophic structures, the study focused on absolute measures of the total volume of hippocampal subdivisions and the total number of neurons.

The report of Onodera et al. [24] demonstrates that the process of increased synaptic plasticity takes place in the hippocampus 3 months after reperfusion. These findings indicate that in subjects who survived transient ischemic episode, the hippocampus undergoes long-term alterations to improve its functional status after damage. Because most studies cover only the period immediately after an ischemic episode, our observations were extended for up to 1 year to investigate long-term changes of the hippocampal neuronal population after ischemic insult.

Section snippets

Materials and methods

Male Wistar rats, weighing 200–250 g, were subjected to 10 min cardiac arrest. All rats belonged to this same breed and were 3 months old at the time of surgery. Animal handling and experimental protocols followed the guidelines of the National Institutes of Health and were approved by the local Commission of Animal Welfare. Global cerebral ischemia was induced according to modified Korpaczew method [4], [25], [26]. Under ether anesthesia, a blunt-end hook, made of steel wire bent at 90°C, was

Results

There were no statistically significant differences in the volume of the hippocampus and its subdivisions between 3-month-old and 15-month-old sham-operated rats (Table 2). The numerical density of neurons in the pyramidal layer of sectors CA2 and CA3 and granular neurons in the dentate gyrus also did not show marked differences between both control groups (Table 3). A statistically significant, age-related increase in neuronal density was found only in the pyramidal layer of sector CA1: from

Discussion

We did not notice statistically significant differences in the total number of neurons between 3- and 15-month-old sham-operated rats either in the pyramidal layer of any cornu Ammonis sector or in the granular layer of the dentate gyrus. The total numbers of pyramidal neurons estimated in our study for sectors CA1 and CA3 are consistent with those obtained by other authors [35], [36]. According to Cassell [37], 12 390 neurons are in the pyramidal layer of sector CA2, about 40% less than the

Acknowledgements

This work was supported in part by funds from the New York State Office of Mental Retardation and Developmental Disabilities and by the Medical Research Centre of the Polish Academy of Sciences, Warsaw. The authors thank Mr Slawomir Januszewski from the Research Medical Center, the Polish Academy of Science, for excellent surgical assistance.

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