Hippocampal neurovascular and hypothalamic–pituitary–adrenal axis alterations in spontaneously type 2 diabetic GK rats

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Abstract

Metabolic and vascular consequences of diabetes mellitus induce several CNS complications. The dentate gyrus of the hippocampus, a well-recognized target for diabetic alterations, is a neurogenic area associated with memory and learning processes. Here, we explored the hippocampal neurogenesis and its microenvironment (astrocytes, vascularisation and glucocorticoid influence) in a spontaneous model of type 2 diabetes, the Goto–Kakizaki rat. The number of proliferative Ki67+ cells and young doublecortin+ neurons was 2-fold higher in the hippocampus from diabetic rats than in normoglycemic control Wistar at 4 months of age. However, there was no difference in cell survival, studied 3 weeks after bromodeoxyuridine administration. Labeling of endothelial cells against von Willebrand factor, demonstrated a 50% decrease in the granular cell layer fractional area covered by blood vessels and a diminished capillary branching in diabetic rats. Finally, Goto–Kakizaki rats exhibited decreased glucocorticoid receptor immunolabeling in CA1, associated with higher corticosteronemia. In conclusion, diabetic rats showed increased cell proliferation and neuronal differentiation without concomitant survival modification. A high proliferation rate, potentially reflecting a compensatory mechanism for neuronal suffering, also exists in various pathological situations. However, endothelial alteration induced by chronic hyperglycemia, hyperleptinemia and insulin resistance and associated with deleterious glucocorticoid effects might impair effective neurogenesis in diabetic Goto–Kakizaki rats.

Introduction

Diabetes mellitus, regardless of the type, is associated with poor performance in cognitive functions, particularly learning and memory, and in complex information processing. These deficits are generally modest. Nevertheless, diabetic patients are known to present increased risk of depression, stroke, dementia and Alzheimer disease (AD) (Biessels et al., 2008, Biessels and Gispen, 2005, Biessels et al., 1994). In diabetes, whether of type 1 (T1D) or type 2 (T2D), hyperglycemia can induce various alterations, such as glucose auto-oxidation, advanced glycation end-products, reactive oxygen species, vascular disturbances and neuroinflammation (Copeland et al., 2008, Ramasamy et al., 2005). As part of the limbic system, the hippocampus, is a crucial CNS target of diabetic alterations in both humans and experimental rodent models (Beauquis et al., 2006, Beauquis et al., 2008a, Jackson-Guilford et al., 2000, Magariños and McEwen, 2000, Ott et al., 1999, Revsin et al., 2005).

In spontaneous and pharmacological mouse models of T1D, we previously described several hippocampal disturbances, including astrogliosis, abnormal neuronal activation, increased oxidative stress together with a marked deficit of dentate gyrus (DG) neurogenesis (Beauquis et al., 2006, Beauquis et al., 2008b). Indeed, the DG is one of the few brain areas, where new neurons are produced during adulthood. The generation of new neurons consists of proliferation, including neuronal fate and specification of progenitors, migration through the granular cell layer (GCL) and maturation and functional integration into neuronal circuits (Abrous et al., 2005, Gage, 2002, Gould and Cameron, 1996, Kempermann et al., 2004, Laplagne et al., 2006, Lie et al., 2004, Ming and Song, 2005, Shors et al., 2002, van Praag et al., 2002). A large proportion of the newly generated cells die after the proliferation step and survival and functional integration are critical endpoints of this process. Newly generated hippocampal cells have been implicated in learning and memory processes (Shors et al., 2002).

Unexpectedly, despite the 90% prevalence of T2D as opposed to the 10% with T1D, there have been much more studies on brain deficits in models of T1D than T2D. The Goto–Kakizaki (GK) rat is a type 2 spontaneous diabetic model, produced by selective inbreeding of Wistar rats using glucose tolerance as a selection index. It provides an interesting polygenic model of diabetes without obesity (Goto et al., 1975, Portha, 2005, Portha et al., 2009). These rats show the main features of the metabolic, hormonal, and vascular disorders usually described in T2D patients. They show mild basal hyperglycemia from weaning onwards (1 month of age), then develop hyperinsulinemia followed by deficient insulin secretion and peripheral insulin resistance later in life. Recently, neuronal abnormalities have been described in 2 spontaneous T2D models. First, Alzheimer-like changes, associated with cortical neurite degeneration and neuronal loss were observed in BBZDR/Wor-rats, which exhibit insulin resistance and hypercholesterolemia (Li et al., 2007). Second, diabetic GK rats showed a reduction of cognitive and exploratory activity, likely caused by learning impairments (Moreira et al., 2007). GK rats had also impaired adult neurogenesis and abnormal proliferation of cultured neural progenitors in response to growth factors (Lang et al., 2008). In addition to hyperglycemia, common features of these 2 spontaneous T2D models are insulin resistance and hypercholesterolemia (Li et al., 2007), which are involved in endothelial dysfunction and chronic vascular disease (Ramasamy et al., 2005). Notably, these metabolic alterations participate in the pathogenesis of atherosclerosis and Alzheimer's disease (Kivipelto et al., 2001, Li et al., 2007) and both represent pathological conditions strongly linked to T2D (Duron and Hanon 2008). Therefore, a vascular origin of neuronal alterations in T2D might be possible.

Moreover, hypothalamic–pituitary–adrenocortical (HPA) axis dysfunction, elevated basal levels of glucocorticoids and impaired stress responses are characteristics of diabetes (Harizi et al., 2007, Stranahan et al., 2008). A link between HPA axis alterations and diabetic “encephalopathy” can be suggested, more precisely involving limbic structures, known to be especially sensitive to stress and related hormones (Reagan et al., 2008, Revsin et al., 2008). Hippocampal neurogenesis is a complex event negatively affected by stress, aging, glucocorticoid administration, inflammation and, as previously mentioned, T1D (Beauquis et al., 2008a, Ekdahl et al., 2003, Eriksson and Wallin, 2004, Gould et al., 2000, Karten et al., 2005, Kempermann, 2002, Kuhn et al., 1996, Mirescu and Gould, 2006). Factors, which positively regulate neurogenesis are: physical exercise, environmental enrichment, steroid hormones, antidepressants, insulin, leptin, growth factors (Jin et al., 2002, Saravia et al., 2007, van Praag et al., 1999) and some pathological situations such as trauma, epilepsy, cerebral ischemia and AD (Mohapel et al., 2004, Taupin, 2006).

New neurons in the subgranular zone of the DG are produced in a microenvironment formed by blood vessels, glial cells, and granular cells with different stages of maturity (Palmer et al., 2000). Therefore, in the present study, we assessed the potential damage inflicted to newborn neurons, glial cells and vessels in 4-month-old type 2 diabetic GK rat hippocampus, compared to age-matched Wistar controls. We measured: a) basal fed glycemia, circulating levels of insulin, leptin and corticosterone; b) the ability for DG cell proliferation measured by Ki67 labeling; c) the differentiation of new neurons by doublecortin (DCX) marker; d) the capacity of newborn cell survival, via detection of 5-bromo-2′-deoxyuridine (BrdU), administered 21 days before killing; e) the astroglial density in the hippocampus using glial fibrillary acidic protein (GFAP) labeling; f) the vascular net of the DG, using the endothelial-specific marker, von Willebrand factor; and g) the hippocampal glucocorticoid receptor level, via specific immunohistochemistry.

Section snippets

Experimental animals

Animal experiments were conducted on 4-month-old male GK and nondiabetic control Wistar rats from the Paris colonies (University Paris-Diderot, France) in accordance with accepted standards of animal care, established by the French National Centre for Scientific Research. Rats were housed under conditions of controlled humidity and temperature (22 ± 1 °C), with lights on from 07:00 am to 07:00 pm.

Metabolic parameters

For metabolic measurement, animals were bled as previously described (Amrani et al., 1998) and then

Circulating parameter disturbances in diabetic GK rats

As shown in Table 1, although being of the same age and sex, diabetic GK rats had a lower body weight than Wistar controls. When examined in the fed state, they exhibited a higher circulating plasma d-glucose concentration than control rats. Plasma insulin concentration was also higher in GK rats than in Wistar controls. However, the insulinogenic index (i.e., the paired ratio between plasma insulin and d-glucose concentration) was significantly lower in GK rats. Serum leptin level was elevated

Discussion

In contrast to T1D, where marked hippocampal astrogliosis, loss of hilar neurons and poor neurogenesis ability have been reproducibly described in different rodent models together with cognitive impairment (Alvarez et al., 2009, Biessels and Gispen, 2005, Revsin et al., 2008, Revsin et al., 2009), little is known about potential brain alterations in T2D. Here, we demonstrated anomalies of hippocampal neurogenesis and its microenvironment in type 2 diabetic 16-week-old GK rats.

Our first finding

Acknowledgments

This work was supported by INSERM (France)–CONICET (Argentina) International Agreement, Naturalia and Biologia (France), National Agency for Science and Technology Promotion (ANPCyT, Argentina, PICT 2006,#1845), University of Buenos Aires Grant (M022, M094, M437) CONICET Grant (PIP 5542). The authors wish to thank Danielle Bailbé for technical assistance. JB is recipient of a Doctoral Fellowship from CONICET.

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