ABSTRACT

Systemic injection of the bacterial endotoxin lipopolysaccharide (LPS) provides a very good means for increasing the release of pro-inflammatory cytokines by circulating monocytes and tissue macrophages. The aim of the present study was to verify the hypothesis that LPS (500 µg/kg b.wt, i.p.) induced cytokine production by macrophages and monocytes activates the pathways causing DNA damage. The observed signal of nick translation from CA1, IB, OB, and CAP regions of hippocampus, and the granule cell layer of cerebellum and the median eminence show more vulnerability to LPS (4 hr exposure) induce DNA damage. The comparative assessment of DNA damage from different brain regions shows that the granule cell layer of cerebellum and median eminence region of the hypothalamus have more DNA damage. These results suggest LPS exert its effects on specific brain cell populations of the central nervous system by release of pro-inflammatory cytokines synthesized by tissue macrophages and circulating monocytes, within accessible structures from the blood.

Key words: endotoxin, biotin, bacteria, ATP, macrophages, monocytes, inflammation, DNA

INTRODUCTION

Exposure of high levels of the bacterial endotoxin lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, often results in septic shock and death. Since the incidence of Gram-negative sepsis has been reported to be in constant progression during the recent years, numerous groups have begun to use systemic LPS administration as a model of infection and inflammation. Systemic LPS injection has been reported to induce changes in blood pressure, osmolarity, pain, oxygen consumption, fever, energy metabolism, multiple organ failure and different changes in the endocrine system, such as activation of the hypothalamic-pituitary-adrenal (HPA) axis (Tilders et al., 1994). Various previous studies have established that the adrenal glucocorticoids damage the hippocampal neurons by affecting the excitatory amino acid system. Adrenal steroids modulate expression of NMDA receptors in the hippocampus (Weiland et al 1995, Bartanusz et al 1995), with chronic glucocorticoids exposure leading to increased expression of NMDA receptor binding in both NR2A and NR2B subunit mRNA levels (Weiland et al 1997). NMDA receptor activation produces concurrent generation of nitric oxide and reactive oxygen species, which initiates the cellular damage (Gunasekar et al., 1995).

Proinflammatory cytokines, including interleukin (IL)-1, Il-6 and tumor necrosis factor (TNF)- are considered to be the main mediators of LPS-induced neuroendocrine changes (Quang et al., 1994, Muramami et al., 1993). Although the primary function of cytokines is aimed at expanding the immunologic mass and activity, cytokines also reach the general circulation and thus trigger different function involved in the neuroendocrine-immune interaction. It is generally believed that the release of pro-inflammatory cytokines from LPS-stimulated monocytes/macrophages, neutrophils and lymphocytes are directly responsible for the immune acute-phase response and the psychopathological outcomes that take place during sepsis (Rosen et al., 1989).

Systemic injection of the bacterial endotoxin LPS provides a very good mean for increasing the release of pro-inflammatory cytokines by circulating monocytes and tissue macrophages. The previous study has shown (Steve et al., 1998) that the LPS exert its effect on mononuclear phagocytes via the cell surface receptor CD14. Further, by in situ hybridization histochemistry with probe specific to CD14 mRNA they have shown the distribution of CD14 mRNA in the arcuate nucleus, dentate gyrus, third ventricle, lateral hypothalamic area, paraventricular nucleus, medial preoptic area and in the granular cell layer of the cerebellum. The present study analyzing the DNA fragmentation (Single nick) by nick translation was undertaken to observe the extent of DNA damage to brain tissue after overactivation of the immune system with intra-peritoneum administration of LPS in rats.

MATERIALS AND METHODS

Experimental animals

Male Wistar rats in the age group of 4-6 months, weighing 150-200g were used in this study. Rats were given an intraperitoneal injection of (500µg/ Kg body weight) LPS (From Escherichia coli, Serotype 055:B5, Sigma, L-2880) in 400µl of pyrogen-free saline or vehicle alone (Saline). Vehicle treated and LPS treated rats were sacrificed by cervical dislocation 4h after the systemic injection. Each experiment was repeated at least 3-4 times.

Nick translation (DNA-polymerase-I mediated biotin-dATP Nick translation (PANT))

30 µm thick fresh frozen coronal sections were cut using s cryostat microtome and were mounted on lysine-coated glass slides and treated in the following manner: 30 min in 4% paraformaldehyde in 50 mM PBS, pH 7.4 at room temperature; 10 min in acetone-ethanol mixture (1:10 at room temperature; 30 min in 1% TritonX-100 in PBS at room temperature; 15 min in 2% (absolute volume) H₂O₂ in tris-buffer (0.5M) at room temperature, and then sections were given 3 washings for 15 min each in PBS at room temperature. Positive controls were given an additional treatment of 200 unit/ml of DNAase prepared in 1x NTB (NTB; Tris HCL (pH 7.8 0.5M), MgCl₂ (50mM), BSA (0.5mg/ml)), 10µl DTT (100mM).

Further the sections were incubated in PANT mixture [For 100µl volume; 40µl autoclaved water, 10µl Nick translation buffer (NTB; Tris HCL (pH 7.8 0.5M), MgCl2 (50mM), BSA (0.5mg/ml)), 10µl DTT (100mM), 8µl nucleotide mixture, 1µl biotin labeled dATP(0.25mM), 1 unit of DNA pol I (Klenow fragment) and 31 µl of autoclaved water] for 3 hr at 37⁰C in a moist chamber.

After giving three washings, 3 min each in PBS, the sections were treated for 5min in PBS-BSA(0.5 mg/ml) at room temperature; 1 hour in Streptavidin-HRP (1:300) solution at room temperature; 3 washings in PBS 3 min each, wash once with tris-saline buffer; 10 min in DAB (0.5 mg/ml) in 0.05% H₂O₂ in tris buffer; wash in tap water; followed by an application of haemotoxylin (5-10 sec), dehydration in alcohol (50, 70,90, 100) and xylene and 1:1 alcohol-xylene, and then mounted with DPX.

The cell count from different regions was observed using a densitometry program (Alphaimage 2000) within a Gel-documentation (AlphaEaseTM, Alpha Innotech Corporation) by taking the fixed size of selected objects in each sample, and the background signal was subtracted by taking a suitable threshold value.

RESULTS

The cell counts from different brain regions are shown in Table 1. The data show the relative vulnerability of different brain regions to LPS induced DNA damage. The CAP region of the dentate gyrus shows DNA fragmentation (40±4) to large extent as compared to other regions of the dentate gyrus like CA1, CA3, IB and the outer blade. Similarly, the cerebellum and hypothalamus show greater DNA fragmentation.

Table 1. Absolute cell number (Mean± S.E.M.) from different brain regions like coronus ammonias (CA1, CA3), inner blade (IB), outer blade (OB), cap region of dentate gyrus (CAP), granule cell layer of cerebellum (CB) and the median eminence region of hypothalamus (HT) from LPS treated rats.

DISCUSSION

Previous studies by Messemer and colleagues (Messemer et al., 1999) has revealed that stimulation of primary bovine glomerular endothelial cells with TNF-alpha or LPS leads to apoptotic cell death as revealed by DNA fragmentation (Ladder formation), cytochrome c efflux, mitochondrial permeability transition, Bak, Bad, Bax, Bcl-2, Bcl-xL protein expression and caspase-3-like protease activity. The above-mentioned study clearly shows that there is severe damage to cellular machinery after immune activation with LPS. Further, studies on rat brain (Nolen et al., 2003) have revealed that lipopolysaccharide (LPS) administration stimulates immune activation, inflammation and deterioration in cell function. Neuronal tissue in cortex and hippocampus are particularly susceptible. The vulnerability of these brain regions has been evaluated by measuring the caspase-3 activation and DNA fragmentation and its relation with stimulation of the mitogen-activated protein kinase, p38 (Nolen et al., 2003).

The previous studies by Steve and his colleagues (Steve et al., 1998) has shown that the mRNA Level of CD14, a surface receptor on circulating monocytes and tissue macrophages, was reached to peak at 4h of post-injection, declined at 6h and reached to basal levels 24h after LPS treatment. Another immunohistochemical study by Iwase and colleagues (Iwase et al., 2000) shows that the iNOS (inducible NOS) and eNOS (endothelial NOS) (located in astrocytes of gray and white matter) stimulated after intraperitoneal injection of LPS. The above-mentioned studies reveal that there is activation of intracellular signaling pathway following systemic treatment with LPS. The present study was undertaken to test the hypothesis the possibility that cytokines released by the activated macrophages and monocytes via blood sepsis to accessible area stimulate the group of pathways those leads to DNA damage in brain tissue. The observed DNA damage accessed by the nick translation study from CA1, IB, OB, and CAP regions of hippocampus (Table 1) shows that these regions of hippocampus are more vulnerable to LPS induced DNA damage. The granular cell layer of the cerebellum and median eminence, which are highly supplied by blood capillaries, show higher DNA damage (Table 1). The observed pattern of tissue damage is similar to that previously observed in various types of brain injury caused by glutamate and hypoglycemia.

The extent of DNA damage as accessed by nick translation (Table 1) from the dentate gyrus suggests that the CAP and inner blade regions are associated with a large number of macrophages, parenchymal and perivascular-associated microglial cell populations which, are vulnerable to LPS-mediated DNA damage before (via CD14 mediated) and after severe sepsis. Similarly, microglial cell populations also heavily occupy the hypothalamus, a primary site of emotion and stress.  The present study shows that the cytokines released by activated macrophages and circulating monocytes play an important role in activation of signaling pathways responsible for DNA damage.

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