Research ReportHypobaric hypoxia damages the hippocampal pyramidal neurons in the rat brain
Introduction
Hypobaric hypoxia encountered at high altitude is known to challenge human cognitive functions (Hale et al., 1996, Liberman et al., 2005). It is reported that chronic HA hypoxia (> 5000 m) resulted in permanent neuronal damage in the human brain (Brierley, 1976), which persisted even after returning to sea level, up to a year or longer (Barke et al., 1993, Cavaletti and Tredici, 1992, Cavaletti and Tredici, 1993). Cognitive functions like learning and memory are adversely affected by exposure to hypobaric hypoxia (Cavaletti and Tredici, 1992). In a similar situation, perinatal hypoxic–ischaemic shock to the brain subsequently led to mental retardation and deficit in cognitive abilities such as learning and memory in rats and human beings alike (Askew, 2002, Arteni et al., 2003, Kumral et al., 2004). Learning and memory functions are an important attribute of the hippocampus (Cervos, 1991, Pulsinelli, 1985).
Though transient hypoxia induces morphological change and permanent neuronal damage in the rat brain (Kirino, 1982), the extent of hypoxic damage is a function of degree and duration of exposure (Hale et al., 1996). It has been demonstrated that severe and chronic (> 5500 m, for 3–4 days) hypoxia/ischemia caused neuronal death in the deep and peripheral brain structures like CA3, CA4 and dentate gyrus of the hippocampus, and thalamus, cerebral cortex, striatum (Freyaldenhoven et al., 1997, Gibson et al., 1981, Naghdi et al., 2003, Smith et al., 1993). Indeed, the deep brain hippocampal neurons were highly susceptible to hypoxic injury (Beal, 1995, Cervos, 1991, Choi, 1996, Pulsinelli, 1985). Cell death in CA1 subfield of the hippocampus has been associated with memory loss without any neurological or neuropathological presentation (Naghdi et al., 2003, Sinden et al., 1997). Transient forebrain ischemia for only 5 min was enough to cause irreversible damage to CA1 pyramidal neurons in the rat brain. However, damages were visible only after 4 days (Kirino, 1982). The CA3 neuronal networks are thought to play crucial role in memory processes and in the generation of synchronous neuronal activities (Hasselmo and Wyble, 1997, Lisman, 1999, Lorincz and Buzsaki, 2000, McNaughton and Morris, 1987, Miles and Wong, 1978). It was shown that hypoxial ischemia resulted in apoptotic death of the neurons in CA3, dentate gyrus and lateral thalamus of the new born rats (Nakajima et al., 2000). From these reports it is conceivable that degenerative changes in the hippocampus could lead to serious cognitive deficits (Barke et al., 1993, Cervos, 1991, Cavaletti and Tredici, 1992, Erecinska and Silver, 1996, Nourhashemi et al., 2000, Smith et al., 1993).
The current understanding of mechanism of action of hypoxic neuronal damage is widely speculative. Nevertheless, a cascade of events are portrayed to be involve in hypoxia induced neuronal injury and damage: depolarization induced Ca2+ entry, the release of excitatory amino acid (e.g. glutamate), intracellular nitric oxide or free radical generation (Horakova et al., 1998), damage to the mitochondrial respiratory enzymes and induction of programmed cell death (Hale et al., 1996). Hypoxia induced free radical production is thought to play significant role in neuronal damage and consequent severe memory deficit (Hale et al., 1996, Horakova et al., 1998). Similarly, many studies suggest that hypoxia induces oxidative stress (Adams, 1975, Askew, 2002, Ilavazhagan et al., 2001, Katz, 1982, Kirino, 1982), leading to oxidative damage of lipids, proteins and nucleic acids (Askew, 2002, Sekhon et al., 2003). Glutamate excitotoxicity is one of the important mechanisms behind hypoxia induced oxidative stress (Hota et al., 2007), which enhances opening of voltage gated Ca2+ channels, thus increasing intracellular calcium level. Ca2+ activates calcium dependent nitric oxide synthase (NOS) to generate nitric oxide (NO), which reacts with superoxide anion (O2.−) to form peroxynitrite (ONOO−) that eventually attacks the membrane lipids, resulting in increased lipid peroxidation (Benveniste et al., 1984, Cassina et al., 2002, Jensen et al., 1991). Excess intracellular Ca2+ also damages the mitochondria by mitochondrial Ca2+ overload which ultimately increases free radical production and decreases ATP level. Large accumulation of free Ca2+ disrupts metabolic function and eventually causes the neuronal death (Mitani et al., 1990). Earlier we have reported that HH causes oxidative stress (Maiti et al., 2006) along with increased intracellular Ca2+ level and decreased mitochondrial membrane potential. We also demonstrated DNA fragmentation due to hypoxia in primary hippocampal culture (Jayalakshmi et al., 2005).
It should be noted that the mechanistic details of the neuronal damage outlined above was largely obtained from ischemia generated either by ligation/occlusion of the carotid artery or chemically induced hypoxia, and was equivalent to anoxic condition. This may not represent the realistic HA environment and hence the true picture of mechanistic details of HA hypoxic neuronal damage may deviate from the current conception. Therefore, we decided to address this issue more precisely. We attempted to closely mimic the hypobaric hypoxia condition at high altitude (6100 m). Moreover, we paid special attention to assess the temporal progress of the neuronal damage on chronic exposure to HH by chosen to expose the animals either for 3 or 7 days. Further, histological analyses were done on the hippocampal CA1 and CA3 regions. We here demonstrated that indeed HA hypobaric hypoxia affected the CA1 and CA3 hippocampal neurons but CA3 region was more vulnerable than CA1 region. It is emphasized that the neuronal damage aggravated with extended exposure time.
Section snippets
Results
The temporal progress of neuronal damage due to hypobaric hypoxia exposure was analysed in the rat brain sections. The data presented here were obtained from two exposure groups, i.e. 3 days or 7 days exposures, both being compared to a normoxic control group (here on called 3 dyHH, 7 dyHH and the control respectively). Both 3 dyHH and 7 dyHH showed significant morphological changes in CA1 and CA3 of the hippocampus as compared to the control (Fig. 1). The exposure groups also displayed more
Discussion
The aim of the present study was to investigate the temporal progress of neuronal damage to the hippocampal neurons in CA1 and CA3 subfields as a consequence of chronic exposure to HH. There are three important findings in this study. First, HH damaged the hippocampal pyramidal neurons. Second, in the hippocampus, CA3 neurons were more vulnerable than CA1 neurons, in chronic HH. Third, the extent of neuronal damage highly corresponded with the duration of hypoxic exposure (Fig. 1, Fig. 2, Fig. 3
Reagents
The following reagents were used in this study. Cresyl violet, para-formaldehyde, absolute alcohol (SIGMA); APO-BrdU TUNEL assay kit (Molecular Probes); Fluoro jade B (Chemicon International); pentobarbital, xylene and paraffin wax (Merck), D.P.X (Ranbaxy).
Animals
Adult male Sprague–Dawley rats (n = 18 and 3 months old) with an average body weight of 225 ± 25 g were used for this study. All rats were maintained in the institute animal house, exposed to 12/12 h light/dark cycles, provided with pellet diet
Acknowledgments
This study was fully supported by the Defence Research and Development Organization, Ministry of Defence, Government of India. We are very much thankful to Prof Birendranath Mallick, School of Life Sciences, Jawaharlal Nehru University for extending the morphometric analysis facility.
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