THE ANATOMICAL RECORD 292:1914–1921 (2009) Structural Features of Ischemic Damage in the Hippocampus ALEXANDER G. NIKONENKO,1 LIDIJA RADENOVIC,2 PAVLE R. ANDJUS,2 1 AND GALYNA G. SKIBO * 1 Bogomoletz Institute of Physiology, Kiev, Ukraine 2 School of Biology, University of Belgrade, Belgrade, Serbia ABSTRACT Cerebral ischemic injury resulting from either focal or global circulatory arrests in the brain is one of the major causes of death and disability in the adult population. The hippocampus, playing important roles in learning and memory, is selectively vulnerable to ischemic insults. Distinct populations of hippocampal neurons are targeted by ischemia and multiple factors, including excitotoxicity, oxidative stress, and inﬂammation, are responsible for their damage and death. Modiﬁcations of synapses occur very early after ischemia, reﬂecting related changes in synaptic transmission. These modiﬁcations structurally relate to spatial patterns formed by synaptic vesicles, geometry of postsynaptic density, and so forth. Ischemiainduced changes of synaptic contacts can be implicated in the mechanisms leading to delayed neuronal death. In this review, we summarize the available data on the structural aspects of ischemic injury of the hippocampus obtained in tissue culture and animal models and discuss pathways of neurodegeneration common for cerebral ischemia and various neurodegeneraC 2009 Wiley-Liss, Inc. tive disorders. Anat Rec, 292:1914–1921, 2009. V Key words: astrocytes; cerebral ischemia; microglia; pyramidal neurons; synapses Cerebral ischemia is caused by major insufﬁciencies in cerebral blood ﬂow. Circulatory collapse due to cardiac arrest leads to global cerebral ischemia, while a focal ischemic lesion may be produced by the occlusion of a major brain artery. Prolonged circulatory deﬁcits induce irreversible changes in the brain tissue inﬂicting the damage and death of neural cells. The primary effect of ischemia is a loss of oxygen and energy substrates, for example, glucose. In contrast to glucose, which has some alternatives, oxygen has no substitute as the ﬁnal electron acceptor in the oxidative phosphorylation pathway, which is why its loss dramatically decreases ATP production and causes an electron leakage generating reactive oxygen species (ROS). Persisting glycolysis leads to intracellular acidiﬁcation further impairing cell functions (Rossi et al., 2007). Soon after ischemia, the malfunction of ATP-dependent processes causes a dramatic change in ion gradients and membrane depolarization associated with a steady rise in extracellular glutamate and other neurotransmitters (Benveniste et al., 1984). As a result, high levels of intracellular Ca2þ trigger cell death. This process, known as excitotoxicity, involves the synergistic action of proteases, ROS, and Ca2þ overload (Choi and Rothman, 1990; Nikonenko et al., 2005). If an ischemic episode is relatively short, then cell death tends to be delayed and selective for neurons; whereas prolonged ischemia leads to a broader and more rapid cellular destruction. Mild ischemia was reported to potentiate synaptic transmission, while more severe ischemic insults were found to suppress it (Hammond et al., 1994; Jourdain et al., 2002). The described events are most prominent in the focus of ischemic injury. The situation is somewhat different in a so-called penumbra, moderately hypoperfused region surrounding the core of injury. Here, energy production is compromised to a lesser extent but ischemia triggers a series of events in the penumbra, which may eventually *Correspondence to: Dr. Galyna G. Skibo, Prof., Department of Cytology, Bogomoletz Institute of Physiology, Bogomoletz street 4, 01024, Kiev, Ukraine. Fax: þ380-44-256-24-42. E-mail: [email protected] Received 18 February 2009; Accepted 9 June 2009 DOI 10.1002/ar.20969 Published online in Wiley InterScience (www.interscience.wiley. com). ISCHEMIC DAMAGE IN THE HIPPOCAMPUS evolve into cell death (Lo, 2008). Some brain regions, including the hippocampus, are more vulnerable to ischemia than others. Here, structural ischemia-related changes can be observed at different levels. To study these changes, the various in vivo (Petito and Pulsinelli, 1984; Ishimaru et al., 2001) and in vitro (Skibo et al., 2000, 2005; Jourdain et al., 2002) models were used. ISCHEMIA-RELATED CHANGES IN NEURONS Cerebral ischemia leads to diverse structural changes in neural cells. Early electron microscopic studies showed transient mitochondrial swelling, disaggregation of polyribosomes, decreases in rough endoplasmic reticulum, and Golgi apparatus in postischemic hippocampal neurons. The recovery of these cells was associated with a marked proliferation of smooth endoplasmic reticulum (Petito and Pulsinelli, 1984). Ischemia causes a dramatic loss of extracellular ﬂuid and concomitant swelling of hippocampal cells (Liu et al., 2001; Kovalenko et al., 2006). These ﬁndings are consistent with diffusion data showing that the volume fraction of intracellular space in the brain tissue which is severely affected by ischemia drops ﬁvefold, from 20% to 4% (Nicholson and Sykova, 1998). Mitochondrial swelling is one of the initial postischemic changes. In mild cases, swollen mitochondria regain their normal shape soon. In severe cases, these organelles demonstrate condensation, increased matrix density, and deposits of an electron-dense material followed by the disintegration of mitochondria (Solenski et al., 2002; Kovalenko et al., 2006). Transient hypoxia slightly decreases active respiration in mitochondria, but together with a rise in intracellular Ca2þ, it causes dramatic effects. Ca2þ should enter the mitochondrial matrix to mediate ischemic injury (Schild et al., 2003). There are alterations in membrane contact points, a rise in intra-mitochondrial Zn2þ level, and activation of large, multi-conductance channels disrupting the functional integrity of the mitochondrial outer membrane in postischemic hippocampal neurons (Bonanni et al., 2006). Cerebral ischemia was shown to inhibit protein synthesis. The increase in the number of so-called stress granules—sites of inactive protein synthesis machinery—was observed within 10–90 min of reperfusion in some areas of the rat hippocampus (Kayali et al., 2005). In the penumbra, protein synthesis gets irreversibly inhibited several hours after ischemia (Zhang et al., 2006). A marked increase in ubiquitin immunoreactivity was shown in postischemic hippocampal neurons (Gubellini et al., 1997). Ubiquitin is used as a tag to label proteins committed to be hydrolyzed, and components of translational complex seem to be heavily ubiquitinated after ischemia (Zhang et al., 2006). In ischemic conditions imbalances between proteases and their inhibitors occur. Ca2þ-dependent cytoplasmic cysteine proteases, calpains, play important roles in cytoskeletal remodeling processes. In neurons, a calpaindependent degradation of the cytoskeleton starts very early after ischemia and fodrin (Yokota et al., 2003), aspectrin (Neumar et al., 2001), MAP2 (Buddle et al., 2003), and other cytoskeletal proteins are being degraded. Another protease—caspase-3 is upregulated mostly in apoptotic neurons (Rami, 2003). In the CA1 area of the postischemic hippocampus caspase-3 overexpression is associated mostly with degenerating pyrami- 1915 dal neurons showing DNA fragmentation. The inhibition of this enzyme signiﬁcantly reduces ischemia-related cell death (Chen et al., 1998). In gerbils, prolonged global cerebral ischemia kills 96% of CA1 neurons by day 4 (Colbourne et al., 1999). Neuronal death in the penumbra occurs at a slower rate taking days to mature (Zhang et al., 2006). Delayed and rapid neuronal death appear to follow a similar sequence of cellular events. The lag between an ischemic episode and neuronal death depends upon the severity and/or duration of ischemia (Rosenblum, 1997). Distinct populations of hippocampal neurons demonstrate differential vulnerability to ischemia (Harry and Lefebvre d’Hellencourt, 2003). Transient cerebral ischemia selectively damaged CA1 pyramidal neurons but left neurons in the CA3 area and dentate gyrus largely intact. Manifestations of cell death in the rat hippocampal CA1 area became quite prominent 1 day after a short ischemic episode. Later, the degeneration of pyramidal cell bodies increased progressively leading to injury and death in 79.5% of CA1 neurons within 7 days (Kovalenko et al., 2006). Hippocampal interneurons are generally more resistant than pyramidal cells to excitotoxic insults possibly because of differences in NMDA receptor expression and/or subunit composition (Avignone et al., 2005). However, some interneurons are more ischemia-sensitive than others, namely, somatostatin (SST)- and neuropeptide Y (NPY)-positive interneurons located in the dentate hilus (Johansen, 1993; Larsson et al., 2001), calretin-positive interneurons in the CA3 area (Hsu et al., 1994), and a small number of interneurons located in the mossy ﬁber layer (Johansen, 1993). SST-positive interneurons have their soma in the hilus and project to the outer molecular layer onto dendrites of dentate granule cells, adjacent to perforant path input. These interneurons, which are very sensitive to excitotoxicity, reduce the likelihood of generating longterm potentiation (LTP) (Tallent, 2007). Calretin-positive CA3 interneurons are characterized by their spiny dendrites and receive more innervation from mossy ﬁbers. Data suggest that the degeneration of CA1 interneurons and CA3 pyramidal cells may occur secondary to the ischemia-related delayed cell death of CA1 pyramidal neurons (Hsu et al., 1994). In contrast, hippocampal GABAergic interneurons, NPY-positive interneurons in the CA1 and CA3 areas as well as parvalbumin-positive cells survive ischemia (Johansen, 1993; Larsson et al., 2001). However, altered GABA neurotransmission may contribute to ischemiarelated neuronal death by enhancing the excitability of CA1 pyramidal cells (Zhan et al., 2006). Mechanisms underlying ischemia-related cell death include necrosis, apoptosis, autophagic death, and hybrid forms of cell death. The distinction between apoptosis and necrosis still relies primarily on morphological criteria (Martin et al., 1998). Cellular necrosis is initiated by the departure from normal physiology and starts with ATP depletion and impairments in membrane permeability. Then nuclear pyknosis follows and the structural integrity of organelles is affected. The resulting release of intracellular debris is accompanied by an inﬂammatory reaction. In contrast, apoptosis is an ordered set of changes in gene expression and protein activity. It starts from chromatin condensation and disintegration of nucleoli. Then dying cells get fragmented forming so-called 1916 NIKONENKO ET AL. apoptotic bodies and cellular debris are phagocytozed by nearby resident cells, typically without generating an acute inﬂammatory response. Apoptotic cells represent a minor fraction of dying postischemic neurons, while the rest follow a necrosislike pathway (Martin et al., 1998; Müller et al., 2004). Upregulation of phospho-c-Jun is viewed as an early sign of apoptosis in CA1 neurons and caspase-3 is upregulated in the majority of apoptotic cells (Müller et al., 2004). The release of cytochrome c from mitochondria to the cytosol is considered to be a critical step in the apoptotic death of CA1 neurons after focal cerebral ischemia (Sugawara et al., 1999). There is some evidence that ischemia-related neuronal death may involve the elements of autophagy—a homeostatic process required for the recycling of proteins and damaged organelles. It was shown that focal ischemia is associated with the enhanced expression of the autophagy regulator Beclin1 and subcellular redistribution of the autophagic marker LC3 (Rami and Kögel, 2008). In addition, it was found that some cells in the penumbra may display hybrid cell death features combining various apoptotic and necrotic alterations: a marked caspase-3 activation, nuclear condensation/ fragmentation, swollen cytoplasm, damaged organelles, and deteriorated membranes (Wei et al., 2004). It is interesting that mild ischemia may stimulate cell proliferation in the subgranular zone of the dentate gyrus. The number of DNA-synthesizing cells expressing a marker of neural stem/progenitor cells was dramatically increased in this region 8 days after ischemia (Yagita et al., 2001). The expression of an inducible nitric oxide synthase (iNOS) seems to be necessary for ischemia-stimulated cell birth, because the latter is not observed after focal cerebral ischemia in mutant mice lacking the iNOS gene (Zhu et al., 2003). Fig. 1. Micrographs of hippocampal tissue in the CA1 area str.radiatum of intact rats (A) and rats subjected to a short episode of transient global cerebral ischemia (B). Scale bar ¼ 200 nm. ISCHEMIA-RELATED CHANGES IN SYNAPSES Synaptic remodeling takes place in hippocampal tissue early after ischemia. A short ischemic episode induces a progressive decrease in synaptic numbers in the rat CA1 area, exceeding 30% at 1 day and 65% at 7 days after the occlusion. Nearly half of the remaining synapses display degenerative features (Kovalenko et al., 2006). In addition, there is a rapid ischemia-related increase in the number of perforated synapses and multiple synapse boutons (Ito et al., 2006; Kovalenko et al., 2006). Perforations are thought to correlate with reactive synaptogenesis, supporting a suggestion that few remaining synapses increase their efﬁcacy in response to ischemia (Jourdain et al., 2002). A signiﬁcant, threefold, increase in the frequency of concave-shaped synapses is observed 1 day after ischemia presumably reﬂecting the enhanced rate of synaptic vesicle exocytosis (Hammond et al., 1994). Consistent with this interpretation, ischemia-related potentiation of excitatory transmission similar to LTP has been reported in vitro (Jourdain et al., 2002). The proportion of perforated synapses dropped almost to control values 7 days after ischemia correlating with the decrease in the frequency of concave-shaped synapses (Kovalenko et al., 2006). It is interesting to observe that the ischemia-related increase in perforated synapse numbers was found in the CA1 area but not in the dentate gyrus af- ter 24 hr of reperfusion (Martone et al., 1999). The number of synapses in the ischemic penumbra was reported to recover 1–12 weeks after ischemia (Ito et al., 2006). Such structural features of a synaptic contact as numbers and spatial distribution of synaptic vesicles (SV) directly relate to the efﬁcacy of synaptic transmission (Fig. 1). SV constitute an integral component of a chemical synapse specializing in the storage of a neurotransmitter and their availability for release determines some presynaptic properties. SV can be divided into three pools characterized by different release-competence: the readily releasable pool (RRP), reserve pool, and resting pool (Sudhof, 2000). RRP vesicles are in close proximity to active zones and have the highest release-competence. Vesicles of the reserve pool are more distant from release sites but can move closer under stimulation. The resting pool contains the organelles that are most distant from active zones and rarely participate in neurotransmitter release. Thus, the position of SV relative to active zones seems to be a reasonable morphological correlate for the probability of neurotransmitter release from a vesicle (Tyler and Pozzo-Miller, 2001). The clustered arrangement of SV is another feature typical of a normally functioning chemical synapse (Kim et al., 2002). Special software was designed to quantify these features in electron micrographs (Fig. 2) (Nikonenko and Skibo, 2004). ISCHEMIC DAMAGE IN THE HIPPOCAMPUS 1917 Fig. 3. Scatterplots visualizing a relation between the distance to the nearest-neighbor vesicle (NND) and shortest distance to the active zone (AZD) for vesicles from control (A) and postischemic synapses (B) (Nikonenko and Skibo, 2004). The ischemia-induced spatial rearrangement of synaptic vesicles can be observed as soon as 15 min after ischemia. Fig. 2. Spatial arrangement of synaptic vesicles relates to the efﬁcacy of synaptic transmission (A). Quantiﬁed by the specialized software the shortest distance from a vesicle to the active zone of a synapse is used as a correlate of a vesicle release-competence (B). Similar formalism is applied to determine spatial proximity of vesicles to each other, that is, their clustering potential (C). Scale bar ¼ 200 nm. In synapses of the rat CA1 area, SV numbers tend to decrease 2 hr after ischemia. This decrease was equal to 20% at 1 day and 44% at 7 days after the insult (Kovalenko et al., 2006). It is consistent with the data showing that cerebral ischemia activates Ca2þ-dependent exocytosis of SV (Hammond et al., 1994). Similarly, SV numbers experienced a 13% drop at 15 min and 25% at 2 hr after an oxygen/glucose deprivation (OGD) episode in vitro. However, one should be cautious when comparing quantities obtained in vivo and in vitro because in the latter case synapses lack proper stimulation, are larger and contain more sparsely distributed SV (Kovalenko et al., 2006). The ischemia-induced spatial rearrangement of SV can be observed in vivo after 15 min of reperfusion, when the average distance from these organelles to active zones increases by 24% (Fig. 3). Later, the value of this parameter gets smaller becoming insigniﬁcantly different from the control 7 days after ischemia. An in vitro ischemia model demonstrates similar changes in the spatial patterns formed by SV. It has to be noted that these changes reﬂect mainly the disappearance of those vesicles which are in close proximity to active zones (Nikonenko and Skibo, 2004; Kovalenko et al., 2006). The inter-vesicle distance may be used as a measure of SV clustering potential. It is of interest that in vivo 1918 NIKONENKO ET AL. this parameter experiences a 10% increase at 15 min (Fig. 3) and 40% increase at 7 days after ischemia. This effect was more pronounced for RRP and part of the reserve pool cluster facing an active zone, the core of a reserve pool cluster being less affected. Despite larger initial values of the parameter for the in vitro ischemia model, its changes were similar in vitro and in vivo. The increase in inter-vesicle spacings indicates the impaired integrity of the spatial clusters of SV and may point to abnormal interactions between SV proteins. The expression of SNAP-25 and synaptophysin implicated in the regulation of synaptic transmission and SV cycling has been modiﬁed following transient ischemia (Ishimaru et al., 2001). A postischemic increase in calpain proteolytic activity and related degradation of cytoskeletal proteins may be responsible for the impairments of SV transport (Sulkowski et al., 2006). The postsynaptic density (PSD) is apposed to a presynaptic active zone being separated from it by a synaptic cleft. PSD is composed of a cytoskeletal frame, in which proteins, including neurotransmitter receptors, ion channels, and adaptor proteins, are anchored. The ischemiarelated activation of proteases results in a partial degradation of certain cross-linker proteins in PSD such as spectrin and MAP2. In addition, unfolding proteins may aggregate here resulting in a more diffuse and irregular PSD structure (Martone et al., 1999). Rapid enlargement of PSD was shown in excitatory spine synapses in the CA1 area in vivo within 24 hr after ischemia, followed by later degeneration of 80% of synaptic contacts. Postsynaptic structures contained numerous vacuoles and lysosomes, and their cytoskeletal elements and membranes demonstrated signs of lysis. A severalfold increase in PSD size was observed 7 days after ischemia. In vitro a transient OGD episode induced changes in PSD similar to those observed in vivo (Kovalenko et al., 2006). It should be stressed that the ischemia-related increase in PSD size was both larger and longer-term in the CA1 area than in the dentate gyrus. Although in the latter, the change was reversed after 4 hr of reperfusion, in the CA1 area, it persisted for at least 24 hr. It was shown earlier that several protein kinases and ATPases are translocated to synapses after ischemia (Hu et al., 1998). The increased amount of material associated with PSD in the postischemic brain can represent denatured and aggregated proteins. PSD proteins in the CA1 area are heavily ubiquitinized, indicating that they are going to degrade (Hu et al., 2000). These changes may cause persistent alteration in synaptic transmission and subsequently contribute to delayed neuronal injury. ISCHEMIA-RELATED CHANGES IN NON-NEURONAL CELLS IN HIPPOCAMPAL TISSUE Despite the fact that neuronal dysfunction is central in the mechanisms of cerebral ischemic injury, other cells also play important roles in the reaction to ischemic insults. Cerebral ischemia causes endothelial cells to form numerous microvilli that may increase microvascular resistance and result in moderate hemodynamic impediments. The injury of endotheliocytes leads to platelet aggregation and formation of occlusive thrombi (Dietrich et al., 1986). Progressive ischemia-related swelling of perivascular astrocytic processes is associated with the narrowing of microvascular lumen (Naganuma, 1990). On the other hand, chronic cerebral ischemia can induce collateral circulation with the formation of new microvessels in ischemic brain regions (Taguchi et al., 2004). The ischemia-related increase in superoxide production induces a vasodilation, abnormal vascular reactivity, and bloodbrain barrier breakdown (Kontos, 2001), which may result in a vasogenic edema (Nagy et al., 2005). Inﬂammatory mechanisms play an important role during the acute phase of cerebral ischemia. Several cytokines and cell adhesion molecules are implicated in early neurological deterioration inﬂuencing infarct volume, while metalloproteinases participate in the development of hemorrhagic transformations (RodriguezYanez and Castillo, 2008). The blockage of interleukin-1 (IL-1) receptors as well as knock-out inactivation of IL-1 gene signiﬁcantly reduced neuronal loss after transient ischemia. There is some evidence that IL-1 may act through the release of nitric oxide by iNOS (Mizushima et al., 2002). It was shown also that interleukin-6 exerts trophic effects on postischemic hippocampal neurons (Matsuda et al., 1996). A metalloproteinase inhibitor signiﬁcantly reduced the ischemia-related injury of these cells. Additionally, ischemic damage was also signiﬁcantly reduced in metalloproteinase-9-deﬁcient mice (Lee et al., 2004). There is a biphasic increase in prostaglandin 2 levels after transient global cerebral ischemia in gerbils, providing the evidence that isoforms of cyclooxygenase are also involved in the progression of ischemic injury (Candelario-Jalil et al., 2003). Glial cells are far less susceptible to ischemic injury than neurons. For example, astrocytes are able to maintain ATP levels longer than neurons; they have fewer ionotropic glutamate receptors and may have better ion buffering and antioxidant capacity. Astrocytes are the only glycogen-storing cells in the brain tissue and can use glycogen as a temporary substitute for glucose in ischemic conditions. Finally, they can release some transmitters, glutamate, D-serine, and adenosine, which have roles in ischemic brain damage (Rossi et al., 2007). Astrocytes provide metabolic and trophic support to neurons and modulate synaptic activity. That is why their impaired functions can critically inﬂuence neuronal survival. Astrocytes respond to ischemia by the increase in the number and size together with elongation of cytoplasmic processes (Kajihara et al., 2001), increase in the expression of glial ﬁbrillary acidic protein (GFAP) of the intermediate ﬁlaments (Rossi et al., 2007), reorganization of their gap junctions supporting a functional syncytium (Li et al., 1998) and transient accumulation of glycogen (Kajihara et al., 2001). Data show that ischemia induces astrocytes to renew the expression of nexin—a potent protease inhibitor with neurite-promoting activity, which is mainly present in the adult olfactory system (Hoffmann et al., 1992). Severe ischemia can induce apoptosis in astrocytes (Yu et al., 2001), however other data suggest that these cells mostly die by a nonapoptotic mechanism (Chu et al., 2007). In gerbils, transient global cerebral ischemia induced an increase in the number of GFAP-positive (astrocytes) and Iba-1-positive (microglia) cells in the CA1 area. This increase was most evident in the str.lacunosum-moleculare, str.moleculare, and hilus 3–7 days after ischemia. On day 28, the reactivity of astrocytes decreased in all layers of the CA1 area. Changes in the intensity of ISCHEMIC DAMAGE IN THE HIPPOCAMPUS 1919 reactive gliosis were consistent with the dynamics of pyramidal cell degeneration. Ameboid, sparsely arranged Iba-1-positive microglial cells were seen in the CA1 area starting one day after the occlusion. On day 3, their numbers had increased more than sixfold in str.pyramidale, but changed little in str.radiatum (Pivneva et al., 2005). Nevertheless, recent evidence indicates that microglia monitor the functional status of synapses and contribute to the subsequent increased turnover of synaptic contacts in the brain tissue affected by ischemia (Wake et al., 2009). In contrast to astrocytes and microglia, ischemia was shown to cause a drop in the number of hippocampal oligodendrocytes (Petito, 1986). RELATION TO NEURODEGENERATIVE DISEASES Despite the fact that cerebral ischemic injury develops much faster than chronic neurodegenerative disorders, they share many common features. Neurodegeneration similar to that observed after cerebral ischemia can also be found in various diseases including Alzheimer’s disease (AD), Parkinson’s disease, and multiple sclerosis. One causative factor typical of all these disorders is an oxidative stress which triggers a cascade of events leading to cell death. Neurodegenerative diseases and cerebral ischemia share the accumulation of oxidative DNA damage (Hill et al., 2008) as well as excitotoxic neuronal degeneration leading to impaired function and ultimate loss of speciﬁc neuronal populations (Arundine and Tymianski, 2003). Interestingly, transient ischemia was shown to cause an abnormal accumulation of b-amyloid peptides in the rat brain tissue (Makinen et al., 2008). The incidence of AD is increased following ischemic episodes, and after chronic hypoxia b-amyloid peptide-mediated increases in L-type Ca2þ channel activity contribute to the Ca2þ impairments seen in AD (Brown et al., 2005). Deposits of a hyperphosphorylated tau protein—a pathological hallmark of many neurodegenerative diseases—accumulate in postischemic neurons in a site-speciﬁc manner (Wen et al., 2004). The development and maturation of neurodegenerative pathologies and ischemic lesions in the brain are characterized by the activation of glial cells and upregulation of inﬂammatory mediators (Akundi et al., 2005; Koistinaho and Koistinaho, 2005). Inﬂammation results in increased nitric oxide formation and nitrosative stress which can inhibit components of the mitochondrial respiratory chain and lead to energy metabolism impairments. This scenario is valid for various neurodegenerative disorders and cerebral ischemic injury (Calabrese et al., 2004). Common features include also Ca2þ-dependent enzymatic proteolysis (Higuchi et al., 2005) and blood-brain barrier breakdown (Willis et al., 2004). Finally, neurodegenerative disorders and ischemic injury share common cell death pathways which basically include a molecularly regulated necrosis (Vanlangenakker et al., 2008) and caspase-mediated apoptosis (Hitomi et al., 2004). CONCLUSIONS It can be concluded that ischemic injury of the hippocampus unrolls at different levels and has both functional and structural implications. The deﬁciency in Fig. 4. A scheme illustrating multiple factors which determine temporal and spatial dynamics of hippocampal ischemic injury. energy metabolism both in neurons and glial cells is an initiating factor. Temporal and spatial dynamics of ischemic injury are dictated by speciﬁc properties of neuronal populations and suggest the action of multiple factors including excitotoxicity, ion imbalance, oxidative stress, inﬂammation, and so forth. Hippocampal tissue reacts against ischemic injury using multiple compensatory mechanisms, for example, synaptic plasticity, activation of resident glial cells, neovascularization, and proliferation of stem cells in the dentate gyrus. The reaction of hippocampal tissue depends on the type (global or focal) and duration of cerebral ischemia. If the latter is relatively short, then cell death tends to be delayed and selective for neurons, whereas longer episodes of ischemia cause broader and faster destructive changes. Neurons die by either necrotic, apoptotic, or autophagic pathways or their death acquires hybrid features. Multiple lines of evidence point to the common nature of neurodegeneration associated with a wide range of pathologies including cerebral ischemia and neurodegenerative disorders; however, it still remains a challenge to study the mechanisms in detail. The investigation of structural aspects of ischemic injury in the hippocampus may provide insight into the pathogenetic mechanisms and help to develop new therapeutic strategies. To date, various agents including calcium and glutamate antagonists, sodium channel blockers, GABAA receptors activators, free radical scavengers, and so forth, have been studied in experimental settings in terms of brain tissue protection against ischemic insults (Kontos, 2001; Lo, 2008). Despite the fact that some studies produced promising results most of them have failed to translate the results into a viable therapy. At the moment, there is no effective therapy to promote the recovery of ischemic brain tissue except for the application of recombinant tissue plasminogen activator which simply degrades the ﬁbrin clot blocking blood ﬂow (Lang and McCullogh, 2008). Reasons for the mentioned therapeutic inefﬁciency are multiple. One could mention a compromised blood ﬂow making difﬁcult on-time delivery of free radical scavengers to the affected region. 1920 NIKONENKO ET AL. Another example deals with a biphasic (injury versus repair) role of some molecular targets in the ischemiaaffected brain tissue (Lo, 2008). However, there are more general aspects limiting the development of effective anti-ischemic therapy. The most important is that successful therapeutic strategy should take into account multiple factors which are in play in the ischemic brain tissue and be targeted at various mechanisms (Fig. 4). For example, our experimental data indicate that in the hippocampus ischemia-related synaptic plasticity occurs well before the degeneration of pyramidal neurons. Thus, from a clinical perspective it would be interesting to investigate the therapeutic potential of agents promoting synaptic plasticity (e.g., estrogens) and evaluate their role in the remodeling of brain tissue injured by ischemia. 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