An ultrastructural study of cell death in the CA1 pyramidal field of the hippocapmus in rats submitted to transient global ischemia followed by reperfusion

Abstract

In the course of ischemia and reperfusion a disruption of release and uptake of excitatory neurotransmitters occurs. This excitotoxicity triggers delayed cell death, a process closely related to mitochondrial physiology and one that shows both apoptotic and necrotic features. The aim of the present study was to use electron microscopy to characterize the cell death of pyramidal cells from the CA1 field of the hippocampus after 10 min of transient global ischemia followed by short reperfusion periods. For this study 25 adult male Wistar rats were used, divided into six groups: 10 min of ischemia, 3, 6, 12 and 24 h of reperfusion and an untouched group. Transient forebrain ischemia was produced using the 4-vessel occlusion method. The pyramidal cells of the CA1 field from rat hippocampus submitted to ischemia exhibited intracellular alterations consistent with a process of degeneration, with varied intensities according to the reperfusion period and bearing both apoptotic and necrotic features. Gradual neuronal and glial modifications allowed for the classification of the degenerative process into three stages: initial, intermediate and final were found. With 3 and 6 h of reperfusion, slight and moderate morphological alterations were seen, such as organelle and cytoplasm edema. Within 12 h of reperfusion, there was an apparent recovery and more ‘intact’ cells could be identified, while 24 h after the event neuronal damage was more severe and cells with disrupted membranes and cell debris were identified. Necrotic-like neurons were found together with some apoptotic bodies with 24 h of reperfusion. Present results support the view that cell death in the CA1 field of rat hippocampus submitted to 10 min of global transient ischemia and early reperfusion times includes both apoptotic and necrotic features, a process referred to as parapoptosis.

Introduction

The brain needs a constant flow of blood, oxygen and glucose to maintain the normal integrity of its different functions. Global transient ischemia results from the complete interruption of cerebral blood flow followed by reperfusion (Zemke et al. 2004).

Brain injury after ischemia involves a complex signaling cascade which comprises depolarization of neurons and glial cells and the release of excitatory amino acids into the extracellular space. Glutamate is an important neurotransmitter in the mammalian central nervous system, which participates in the excitatory amino-acid mediated neuronal degeneration known as excitotoxicity (Olney, 1969).

Glutamate released to the presynaptic terminal acts on postsynaptic metabotropic and ionotropic receptors. Activation of ionotropic receptors, mainly NMDA, is involved in many diseases, including gray and white matter excitotoxicity (Li & Stys, 2000; Matute et al. 2002; Waxman & Lynch, 2005; Micu et al. 2006). Receptor activation induces elevation in the concentration of intracellular ions, especially Ca⁺² and Na⁺ (Mattson et al. 2000; Zemke et al. 2004). Under pathological conditions, excessive stimulation of NMDA receptors may cause alterations in the ionic gradient which induces toxic cellular swelling and triggers many downstream neurotoxic cascades, including the uncoupling of mitochondrial electron transfer from ATP synthesis and the overstimulation of enzymes that damage the cell, such as lipases, proteases, phosphatases and nucleases (Tymianski & Tator, 1996; Mattson et al. 2000; Arundine & Tymianski, 2004; Waxman & Lynch, 2005).

The 4-vessel occlusion (4VO) method of inducing ischemia involves cauterization of both vertebral arteries plus transient occlusion of the carotids and results in significant reduction of the cerebral blood flow (90% or more), producing approximately 90% of neuronal death 24 or 48 h after the ischemic insult (Pulsinelli & Brierley, 1979; Nunn et al. 1994; Frassetto et al. 2000). The hippocampus has been extensively utilized to evaluate neuronal death after cerebral ischemia due to its simple arrangement and homogeneous pattern of damage within each region. The dorsal hippocampus, and particularly the CA1 pyramidal cells, show a significant degree of vulnerability to transient ischemic damage of short duration (≤ 15 min) of 4VO (Kirino et al. 1984; Nunn et al. 1994; Nitatori et al. 1995).

Which cellular death pathway is taken after global transient ischemia remains an open question. Excitotoxic cell death is closely related to mitochondrial physiology and may involve either apoptotic or necrotic features. The pattern of neuronal hippocampal death after 4VO ischemia can be evaluated by morphological modifications visualized both at optical and electronic microscopic levels and appears to be intimately related to the quality and duration of the aggressor stimuli, animal age, nutritional conditions and experimental model employed (Kirino et al. 1984; Bicknell & Cohen, 1995; Sheldon et al. 2001; Blomgren et al. 2003; Ruan et al. 2003; Ouyang & Giffard, 2004; Winkelmann et al. 2006).

Apoptosis is a process that requires energy and is present both in physiological and neuropathological conditions. Several events characterize apoptosis, including a decrease in nuclear size, nuclear chromatin aggregation, nucleolus disintegration, cytoplasmic condensation and maintenance of the organelle integrity. In the final stages, apoptotic bodies are formed and then quickly eliminated by phagocytic cells (Martin et al. 1998; Roy & Sapolsky, 1999; Hou & Macmanus, 2002; Rami, 2003). On the other hand, cellular necrosis only occurs in pathological circumstances and comprises modifications of cellular volume with disruption of the plasma membrane. In contrast to apoptosis, necrosis is associated with adjacent tissue inflammation (Kerr et al. 1995; Martin et al. 1998; Rami, 2003; Ruan et al. 2003).

Previous studies have shown that cell death after ischemia involves characteristics of necrosis (Colboune et al. 1999; Winkelmann et al. 2006), of apoptosis (Zeng & Xu, 2000) and a combination of both processes (Nitatori et al. 1995; Ruan et al. 2003), when cellular structure was evaluated 24 h or more after the event.

We have recently suggested that cell death occurring after forebrain ischemia be classified into three phases, according to the degree of cell degeneration. Briefly, the initial phase is characterized by intact membranes, minimum modification of the mitochondria, slight dilatation of rough endoplasmatic reticulum (RER) and Golgi complex (G) and the presence of small chromatin clumps. Neurons in the intermediate phase exhibit increased heterochromatin, advanced edematous cytoplasm and fragmented RER and G. In the final stage there is a disruption of membranes, intense morphological alteration of organelles and the presence of structures containing cellular debris. Intact neurons are observed among these degenerate cells (Winkelmann et al. 2006); it might be that these seemingly intact cells are developing delayed cell death, as characterized by Pulsinelli et al. (1982).

Considering that the mechanisms of post-ischemic neuronal death remain unclear, the aim of the present study was to characterize death of pyramidal cells from the CA1 field of the hippocampus after 10 min of transitory global ischemia followed by short reperfusion periods (≤ 24 h), by means of ultrastructural analysis using electron microscopy. We hypothesize that both necrotic and apoptotic characteristics will be found.

Materials and methods

Animals

For this study 25 adult male Wistar rats weighing between 280 and 320 g from the UFRGS Animal House (ICBS, Universidade Federal do Rio Grande do Sul) were used. The animals were kept under a constant 12:12 h light-dark cycle at a room temperature of 22 ± 1 °C and maintained with food and water ad libitum. The Brazilian Laws and the National Institute of Health Guide for Animal Care and Use of Laboratory Animals were strictly followed.

Experimental groups

The animals were divided into five groups, each with five animals. Twenty animals received 10 min of ischemia, and were divided into four groups receiving 3, 6, 12 and 24 h of reperfusion respectively, and a control group consisting of intact (untouched) animals.

Ischemic surgical technique

Transient forebrain ischemia was produced using the 4-vessel occlusion method, with minor modifications (Pulsinelli & Brierley, 1979; Netto et al. 1993; Winkelmann et al. 2006). Briefly, on the first day of the experiment animals were anaesthetized with Halothane (Cristália, São Paulo, Brazil), mixed with breathing air, placed on a rat operating table and an occluding device (silastic ties) was loosely placed around each carotid artery to allow subsequent occlusion of these vessels with minimal mechanical disturbance. The animals were then placed on a stereotaxic frame and the vertebral arteries were eletrocoagulated through the alar foraminae of the first cervical vertebrae. On the following day, rats were restrained and the occluding device was activated for 10 min, producing a temporary occlusion of both common carotid arteries and so completing the 4-vessel occlusion (carotid + vertebral). Animals that did not lose the righting reflex or that convulsed during the ischemic insult were not used in the experiments. In all experiments, the body temperature was maintained at 37 °C during the ischemic insult by the use of a homeothermic blanket (Letica, Barcebna, Spain). After the surgery, animals were returned to their cages with free access to water and standard rat chow. The control group was composed of intact animals.

Electron microscopy

After either 3, 6, 12 or 24 h of reperfusion, the animals were re-anaesthetized with 0.1 mL 100 g⁻¹ of sodium thiopental (Cristália) and injected with 1000 UI heparin (Cristália). After that, they were transcardially perfused through the left ventricle, using a peristaltic pump (Control Company, São Paulo, Brazil; 20 mL min⁻¹) with 200 mL of saline solution followed by 100 mL of fixative solution composed of 2.5% glutaraldehyde (Sigma, St Louis, MO, USA) and 2% paraformaldehyde (Reagen, Rio de Janeiro, Brazil) in 0.1 M phosphate buffer (PB) pH 7.4 at room temperature. Then the brain was removed and immersed overnight in the same fixative solution.

Coronal sections (100 µm) of the brain were obtained using a vibratome (Leica, Wetzlar, Germany) and postfixed in the same fixative solution for at least 1 h. Then the sections were washed in phosphate solution and postfixed in 1% osmium tetroxide (Sigma) for 1 h at room temperature. The material was dehydrated in ascending graded series of alcohol and propylene oxide (Electron Microscopy Sciences, Hatfield, PA, USA), embedded in araldite (Durcupan ACM, Fluka, Buchs, Switzerland) and maintained in a vacuum for 24 h; after that they were put onto slides with resin and polymerized for 48 h at 60 °C. Areas containing the CA1 field of the dorsal hippocampus were selected (Fig. 1) and removed from the sections, employing a stereomicroscope (Wild, Heerbrugg, Switzerland). The selected CA1 field sections were glued onto a resin block and newly polymerized for 48 h at 60 °C. Semithin sections (1 µm) were obtained using an ultramicrotome (M T 6000-XL, RMC, Tucson, USA) and stained with 1% toluidine blue diluted in 1% sodium tetraborate to identify the CA1 pyramidal neurons (Rodrigo et al. 1996). Then ultrathin sections (80 nm) were obtained with the same ultramicrotome and stained with 2% lead citrate (Merck, Darmstadt, Germany) and 1% uranyl acetate (Merck) (Reynolds, 1963), and then examined using a transmission electron microscope (JEM 1200 EXII, Akashima, Japan, CME-UFRGS).

Fig. 1.

Photomicrography of the CA1 field of rat hippocampus. The boxed area indicates the region analyzed. CA, Cornus Ammonis. Scale bar: 300 µm.

Results

The CA1 field pyramidal cells from rat hippocampus submitted to ischemia exhibited intracellular alterations consistent with a degenerative process, and the ultrastructural features of these alterations varied in intensity according to the reperfusion period.

Control group

In the control group (Fig. 2), the pyramidal neurons had a pale round or oval nucleus (Fig. 2A) surrounded by cytoplasm with mitochondria, smooth endoplasmic reticulum (SER), G, RER, free ribosomes and polysomes, lysosomes, neurotubules and neurofilaments (Fig. 2B). Blood capillaries were surrounded by thin astrocytic end feet and showed endothelial cells with euchromatic nuclei, tight junctions between the adjacent plasma membranes and an absence of transendothelial vesicles (Fig. 2C).

Fig. 2.

Electron micrographs from CA1 field of control animals. (A) Euchromatic pyramidal neuron, nucleus (N), nucleolus (nu), plasma membrane (double arrow) and nuclear membrane (arrow). (B) Detail of the cytoplasm, showing Golgi complex (G), rough endoplasmic reticulum (RER), free ribosomes (R) and mitochondria (M). (C) Blood capillary (BV) with preserved lumen, surrounded by astrocytic end feet (EF – asterisks), showing an heterochromatic endothelial nucleus (EC). Scale bar: A: 2 µm; B: 500 nm; C: 1 µm.

10 min of ischemia followed by 3 h of reperfusion

In comparison with the control animals, the rats submitted to global transitory ischemia followed by 3 h of reperfusion showed slight ultrastructural alterations (Fig. 3). Most neurons exhibited a pale round or oval nuclei and intact nucleoli, sometimes localized in eccentric positions. Plasma and nuclear membranes remained intact, as did the neurofilaments and mitochondria, which maintained their normal appearance, with recognizable cristae. However, many empty spaces were found inside the large electron lucent nuclei (Fig. 3A). Initial cytoplasmic vacuolization was also detected. While most neurons had slightly dilated RER and G, some cells displayed more significant edema and morphological modifications in their organelles (Fig. 3B). Sometimes polysomes were disassociated, displaying desegregated ribosomes, giving the cytoplasm a dark aspect. Pleomorphic lysosomes with a dense content were evident when compared with control animals and some electron dense neurons appeared among clear cells. These cells displayed dilated RER, though ribosomes persisted against the cisternae. In addition, neurons with electron lucid cytoplasm also exhibited some chromatin clumps, scanty organelles, mitochondria and altered SER.

Fig. 3.

Electron micrographs of CA1 field submitted to 10 min of ischemia followed by 3 h of reperfusion. (A) Euchromatic nucleus (N), nuclear membrane preserved (arrow), intact plasma membrane (double arrow), glial cell – microglia (GC), blood vessel (BV). (B) Cellular debris with edematous and dark mitochondrion (M), Golgi complex (G) and rough endoplasmic reticulum (RER) with ribosomes against them. (C) Detail of the electron micrograph A illustrating a blood vessel (BV) with diminished lumen, endothelial cell (EC), edematous astrocytic end feet (EF) surrounding the capillary (asterisks) with decreased glycogen granules. Scale bar: A: 2 µm; B, C: 1 µm.

The oligodendrocytes had a normal dense aspect. Nuclei were irregularly shaped and RER were more dilated than in normal conditions, however ribosomes remained attached to their cisternae. Several astrocytes showed nuclei with irregular shapes, pale and edematous cytoplasm, relatively sparse organelles, some pleomorphic lysosomes and glycogen granules. Bundles of intermediate filaments were distributed throughout the perikaryon, but within the processes the distribution was looser than that found in the control animals. Some astrocytic end feet surrounding the blood capillaries were swollen, showing electron lucid cytoplasm with some vacuoles, glycogen granules and disassociated intermediate filaments. The capillary lumen was also modified, showing a reduced diameter due to folding of the vessel wall (Fig. 3C).

10 min of ischemia followed by 6 h of reperfusion

In animals submitted to 10 min of ischemic insult followed by 6 h of reperfusion, the neuronal changes were more evident than in the 3 h reperfusion group (Figs 4 and 5). Several neurons exhibited wrinkled nuclei, with angular shapes (Fig. 4A) and the persistence of nuclear vacuoles. Chromatin clumps appeared against the inner aspect of the nuclear membrane and dispersed into the karyoplasms. While the nuclear membrane was usually intact, at times it was arranged in deep folds that extended across almost all the nuclear diameter. Though, the nucleoli frequently appeared normal, they sometimes displayed shape modifications and an electron lucid appearance, with predominance of the pars fibrosa(Fig. 4B). RER cisternae lacked ribosomes, were dilated and several were in the process of forming vacuoles (Fig. 4C,D). There was an increase in the numbers of free ribosomes located throughout the cytoplasmatic matrix and some polysomes were disassociated, while the G was fragmented (Fig. 4D). Some mitochondria were swollen and presented structural disorder with a vacuolated aspect and scanty cristae, while others appeared intact. The plasma membrane was usually found to be intact (Fig. 4D). Numerous cytoplasmic vacuoles containing cellular debris were identified (Fig. 5A,B).

Fig. 4.

Electron micrographs of CA1 field submitted to 10 min of ischemia followed by 6 h of reperfusion. (A) Pyramidal cell exhibiting a heterochromatic nucleus (N) with angular shape (narrow), nucleolus (nu) with predominance of pars fibrosa. (B) Pyramidal neuron showing nucleus (N) with some indentations and preserved nuclear membrane (arrow), nucleolus in eccentric position (nu), with predominance of pars fibrosa, intranuclear vacuoles (V). Chromatin clumps against the inner nuclear membrane (double arrow). (C) Detail of electron micrograph A heterochromatic nucleus (N), dilated rough endoplasmic reticulum (RER), slight modifications in Golgi complex (G) and in mitochondrion (M). (D) Detail of electron micrograph A, intact nuclear membrane (arrow), dilated rough endoplasmic reticulum (RER), edematous Golgi complex (G), mitochondria with vacuoled aspect (M), preserved plasma membrane (double arrow). Scale bar: A, B, C, D: 1 µm.

Fig. 5.

Electron micrographs of CA1 field submitted to 10 min of ischemia followed by 6 h of reperfusion. (A) Neuron in final stage of degeneration displaying cellular debris (CD), glial cell (GC). (B) Other cell in final stage of degeneration, showing cellular debris (CD). (C) Detail of electron micrograph A, pyknotic nucleus, edematous mitochondria (M), rough endoplasmic reticulum (RER) with some ribosomes. (D) Detail of electron micrograph B, pyknotic nucleus, swollen and dark mitochondrion (M), fragmented Golgi complex (G) and rough endoplasmic reticulum cisternae (RER) with ribosomes. Scale bar: A, B, C, D: 1 µm.

Some neurons in the intermediate stage of the degenerative process exhibited increased heterochromatin at the initial stage of pyknosis and very pale and edematous cytoplasm with fragmented RER and G (Fig. 5C,D). Small Nissl bodies persisted in the cytoplasm where mitochondria were of different sizes, with dilated spaces in the matrix and no identifiable cristae.

The blood capillaries were surrounded by astrocytic end feet with significant edema and loose intermediate filaments, with a fuzzy aspect. The endothelial cells showed several transendothelial vesicles, numerous microfolds on the luminal side, intact basal lamina and tight junctions.

10 min of ischemia followed by 12 h of reperfusion

In animals submitted to 10 min of ischemia and 12 h of reperfusion, neurons in different stages of degeneration were observed (Figs 6 and 7). Several neurons retained their normal aspect with little modification (Fig. 6A). This may suggest recuperation, since they displayed more severe alterations in the 3 and 6 h of reperfusion groups. These neurons had large round nuclei, clear karyoplasm, intact nucleoli and slight organelle modification. Nevertheless, other cells exhibited irregularly shaped nuclei, numerous nuclear indentations (Fig. 6B), more numerous bundles of nuclear filaments and chromatin clumps against the double nuclear envelope. Where vacuolization persisted in some neurons (Fig. 6C), the nucleolus, located eccentrically, was more electron lucid and there was a predominance of the pars fibrosa. In some cases, the nuclear membranes were disrupted. Some mitochondria had identifiable cristae as well as outer and inner membranes. Other mitochondria were edematous displaying an electron dense aspect and a loss of matrix structure (Fig. 6D). RER and G presented greater cisternal dilation giving rise to cytoplasmic vacuoles (Fig. 6E). Some ribosomes remained against the RER cisternae; filaments, microtubules, pleomorphic lysosomes, as well as the polysomes were apparently intact (Fig. 6F). Though the plasma membrane also remained intact in most neurons, in more advanced stages of degeneration it was not detected. In such cases, the vacuoles in the neuronal cytoplasm and neuropilar region showed membrane agglomeration or myelin-like figures. Empty spaces containing cellular debris were also identified (Fig. 6F).

Fig. 6.

Electron micrographs of CA1 field submitted to 10 min of ischemia followed by 12 h of reperfusion. (A) Pyramidal cells with minor ultrastructural modifications with dendritic process (D), intact nuclear (arrow) and plasma membranes (double arrow). (B) Dark pyramidal neuron, nucleus (N), nuclear indentations (arrow), nucleolus (nu) in eccentric position and with predominance of the pars fibrosa, compacted cytoplasm (asterisk), preserved plasma membrane (open arrow). (C) Neuron with hetechromatic nucleus (N and arrow), neurofilaments (Nf), rough endoplasmic reticulum residues (RER) with several ribosomes. (D) Detail of electron micrograph C showing a swollen mitochondrion (M), cytoplasm with scanty organelles, preserved nuclear membrane (arrow). (E) Pyramidal cell cytoplasm displaying high dilated rough endoplasmic reticulum (RER), little modification in mitochondrion (M), lysosomes (Ly). (F) Neuron in final stage of degeneration showing a nucleus (N) with chromatin aggregates against the nuclear envelope, edematous cytoplasm with sparse organelles, neurofilaments (Nf) and rough endoplasmic reticulum (RER) with some ribosomes, dark and swollen mitochondria (M). Scale bar: A, B: 2 µm; C, F: 1 µm; D, E: 200 nm.

Fig. 7.

Electron micrographs of CA1 field submitted to 10 min of ischemia followed by 12 h of reperfusion. (A) Blood vessel (BV) with preserved lumen, endothelial cell with euchromatic nucleus (EC), edematous astrocytic end feet (EF) without glycogen granules, pericyte (GC). (B) Oligodendrocyte cell (OC) with dense cytoplasm and heterochromatic nucleus. Scale bar: A, B: 500 nm.

The astrocytes showed increased ultrastructural alterations. In some blood capillaries the luminal space and the basal lamina were preserved, but were surrounded by edematous astrocytic end feet in which glycogen granules and transendothelial vesicles were absent (Fig. 7A). Microglial cells and oligodendrocytes exhibited no significant ultrastructural modifications (Fig. 7B).

10 min of ischemia followed by 24 h of reperfusion

Animals submitted to 10 min of ischemia and 24 h of reperfusion displayed more severe signs of the degenerative process. In only a few neurons was the ultrastructure retained intact (Figs 8 and 9).

Fig. 8.

Electron micrographs of CA1 field submitted to 10 min of ischemia followed by 24 h of reperfusion. (A) Pyramidal neuron with heterochromatic nucleus (N) with edematous and vacuoled cytoplasm with scanty organelles (asterisk), glial cell (GC). (B) Detail of electron micrograph A, showing the cytoplasm with fragments of rough endoplasmic reticulum (RER) and neurofilaments (Nf) with fuzzy aspect, dark mitochondrion (M) and absent plasma membrane. (C) Detail of the pyramidal cell, showing the nucleus (N), dilated rough endoplasmic reticulum (RER) and Golgi complex (G), vacuoled mitochondria (M), intact nuclear (arrow) and plasma (open arrow) membranes. (D) Detail of neuron showing nucleus (N), dark and swollen mitochondria (M), dilated rough endoplasmic reticulum (RER) and Golgi complex (G), lysosomes (Ly), intact nuclear membrane (arrow). (E) Pyramidal cell cytoplasm with pleomorphic lysosomes (Ly), scanty dilated rough endoplasmic reticulum (RER), preserved nuclear (arrow) and plasma membranes (open arrow), cytoplasm of other cell with edematous aspect, neurofilaments (Nf) and dark vacuoled mitochondrion (M). Scale bar: A, B: 1 µm; C: 200 nm; D, E: 500 nm.

Fig. 9.

Electron micrographs of CA1 field submitted to 10 min of ischemia followed by 24 h of reperfusion. (A) Neuronal cytoplasm showing some apoptotic bodies (AB), rough endoplasmic reticulum (RER) and polysomes. (B) Detail of electron micrograph A showing an apoptotic body (AB) with dense content surrounded by a membrane and with apparently intact mitochondrion (M) and rough endoplasmic reticulum (RER). (C) Blood vessel (BV) with diminished lumen, endothelial cell (EC) and high edematous astrocytic end feet (EF) surrounding the capillary (asterisks). Scale bar: A, B: 500 nm; C: 2 µm.

In some cases, neurons in the initial stage of degeneration exhibited large euchromatic nuclei; in others, nuclei with small accumulations of chromatin (Fig. 8A,B). More numerous bundles of nuclear filaments were observed and the G were dilated, though they kept their parallel arrangement (Fig. 8C). There was some dilation of the RER, but the ribosomes remained attached to their cisternae (Fig. 8D). Also, in some neurons the plasma and nuclear membranes remained intact.

Most neurons showed more severe signs of degeneration than at other reperfusion times, displaying more nuclear indentations, shape irregularity, chromatin clumps and chromatin agglomerations against the nuclear envelope. The cytoplasm was often very pale, with an edematous, vacuolated aspect and scantly organelles (Fig. 8A). Though most nuclear membranes were disrupted, the plasma membrane remained intact. The vast majority of mitochondria had an altered aspect, in which the matrix was darkened and the cristae were not visible (Fig. 8B). The SER, G and RER were extremely swollen, and there was a total absence of ribosomes against the RER cisternae, forming vacuoles throughout the perinuclear cytoplasm (Fig. 8E). Pleomorphic lysosomes and disassociated polyribosomes were also identified.

Intense disorganization of the neuropilar region was noted in the analyzed animals. Organized structures containing cellular debris were observed in the neuropilar region; some had a dark or electron dense aspect. Moreover, degenerative signs such as membrane agglomeration and myelin-like figures were seen more frequently than in the 12 h reperfusion group.

In animals submitted to ischemic insult and 24 h reperfusion, some structures with apoptotic features were found: chromatin aggregated into large round clumps; preserved RER, with ribosomes against their cisternae and relatively intact mitochondria. In some cases, the chromatin clumps were round and surrounded by a membrane unit, resembling apoptotic bodies (Fig. 9A,B).

The astrocytic end feet that surrounded the blood capillaries were excessively swollen, with numerous vacuoles in the electron lucid cytoplasm, and occasionally there was an absence of glycogen granules. The vascular wall of the capillaries displayed folds which resulted in intense reduction of the luminal side. The basal lamina and the tight junctions were preserved and some transendothelial vesicles were also identified (Fig. 9C).

Discussion

Alterations in the ultrastructural morphology of the brain tissue were observed in all analyzed experimental animals. Since there were different degrees of pathological modifications, which in part could be related with the reperfusion time, it is possible to classify the degenerative process into three phases: initial, intermediate and final (as described by Winkelmann et al. 2006, for longer reperfusion times). Thus, in the same reperfusion period we were able to identify neurons in different stages of degeneration and not all the nerve cells displayed signs of degeneration.

While there is evidence in the literature to assert that neuronal death in the CA1 subfield of gerbil hippocampus submitted to 5 min of ischemia only occurs 24 h after the insult (Nitatori et al. 1995), we observed slight structural alterations such as swollen organelles and the formation of nuclear vacuoles at earlier periods (3 and 6 h of reperfusion, that could be classified as the initial and intermediate stages of degeneration, respectively).

There is no consensus about the classification of different types of programmed cellular death (PCD). Analysis of the nuclear and mitochondrial morphology makes it possible to categorize PCD as classical apoptosis, apoptosis-like or necrosis-like processes (Krantic et al. 2005). Mitochondria are known to be involved in both necrotic and apoptotic pathways and the type of cellular death depends on the severity of the insult (Matute et al. 2002; Ouyang & Giffard, 2004; Winkelmann et al. 2006). In many cases, ‘severe’ brain ischemia results in complete mitochondrial dysfunction, which hampers adenosine triphosphate (ATP) production, ensuring necrotic cell death (Chan, 2004; Moro et al. 2005).

Since the ischemic insult was the same to all groups we can infer that the prevalence of necrotic characteristics in the short reperfusion periods was due to the immediate energy failure which would disable the apoptotic pathway. In these stages severe physiological modifications probably occurred in the mitochondria, although they were not seen to the same degree at the morphological level. Given that apoptotic death needs energy in order that cellular modifications such as cytoskeletal proteolysis and DNA alteration (Roy & Sapolsky, 1999) take place, continued PCD would be impossible if the mitochondrial physiology were impaired. In this case, characteristics of necrosis will appear (Martin et al. 1998; Lipton, 1999).

Several necrotic features continued to be present at 6 h after ischemia (Fig. 5A–D); however decreases in the size of the nucleus and nuclear membrane presenting deep folds were seen. These features correspond to the apoptotic process in which precocious desegregation of these structures occurs (Wyllie et al. 1980; Martin et al. 1998). Thus, the nuclear contraction and the formation of small chromatin clumps with complete membranes could indicate that the cell is dying in a ‘clean way’, without inflammation and the secondary harmful effects of cytokines (Kerr et al. 1995; Roy & Sapolsky, 1999; Rami, 2003; Ruan et al. 2003).

In contrast to our expectations, rats receiving 12 h of reperfusion exhibited cells with apparent morphological recuperation. As in the case of shorter reperfusion times, neurons with varied morphological alterations were identified, but there was a notable predominance of apparently intact neurons. In the 24 h reperfusion group, neurons in the intermediate and final stages of degeneration were more prevalent (Fig. 8A,B). In these two reperfusion times (12 and 24 h) some cells exhibited features frequently described in apoptosis. However, when high magnification was used, it was possible to note that these same cells did not display the ‘classic features of apoptosis’ (Sheldon et al. 2001). On the other hand, typical characteristics of necrotic death (Martin et al. 1998; Briones et al. 2004) were present in both early and later reperfusion times (Figs 3A,B and 6F).

We can suggest that at 12 h of reperfusion, neurons changed the cell death pathway from necrosis to PCD, as demonstrated in Fig. 6E (intact appearance of mitochondria). Moreover, filaments and microtubules were apparently intact, which indicates apoptosis (Fig. 6F) (Bursch, 2004; Winkelmann et al. 2006). As PCD is a process that depends on mitochondrial integrity (Martin et al. 1998) and sufficient production of ATP (Weishaupt et al. 2003), apoptosis would be possible with the restoration of energy. This might explain the appearance of signs of apoptosis in the neurons at 24 h of reperfusion together with apparently intact organelles.

Altogether, our findings could mean that as soon as 3 h after ischemia, while ATP remains available, the cell initiates a process of cellular degeneration similar to apoptosis. However, over prolonged reperfusion times (6 h), with impairment of the blood perfusion, mitochondrial failure and energy collapse, it becomes impossible to complete the process of programmed cellular death. In which case, the pattern of cell death may change, and a degenerative process similar to necrosis may develop (Weishaupt et al. 2003; Yuan et al. 2003). At longer reperfusion times (12 and 24 h) with a probable restoration of energy it may be possible that some cells died by apoptosis while others continued the necrotic process.

Glial cells displayed an increase in the degree of the ultrastrucutural alterations in relation to the length of the reperfusion time. In all animals, modifications were identified to the astrocytic end feet, whose edema reduced the vascular lumen, and probably hampered blood perfusion (Figs 3C and 9C). In global ischemia, there is often marked hypoperfusion, which is extreme between 6 and 24 h after the insult, with blood flow between 30 and 50% of normal conditions (Kagstrom et al. 1983; Lipton, 1999). Morphological alterations in capillary diameters have been described after ischemia, showing that they can decrease significantly 3 to 24 h after ischemia, return to baseline level and become significantly larger 28 days after the surgery (Taguchi et al. 2004).

Due to the diversity of the morphological modifications, which sometimes indicate apoptosis and, at other time points, necrosis (Barth et al. 2002; Liu et al. 2004; Dyuyniewska et al. 2005), many scientists recognize the possibility that ischemic neurons undergo a process that would be a ‘continuum’ between apoptosis and necrosis, referred to as ‘parapoptosis’ (Martin et al. 1998; Roy & Sapolsky, 1999; Sheldon et al. 2001; Hou & Macmanus, 2002). The present findings indicate that ischemic neuronal death during early reperfusion times is due to a process of ‘parapoptosis’, with a prevalence of necrotic characteristics (Muller et al. 2004).