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Behavioral and ultrastructural changes induced by chronic neuroinflammation in young rats
Brain Research 859 Ž2000. 157–166 www.elsevier.comrlocaterbres
Research report
Behavioral and ultrastructural changes induced by chronic neuroinflammation in young rats Beatrice Hauss-Wegrzyniak a , Maria G. Vannucchi b , Gary L. Wenk a
a, )
Arizona Research Laboratories, DiÕision of Neural Systems, Memory and Aging, 350 Life Sciences North Building, UniÕersity of Arizona, Tucson, AZ 85724, USA b Department of Anatomy and Histology, UniÕersity of Florence, Florence, Italy Accepted 4 January 2000
Abstract We investigated the ultrastructural, immunohistochemical, biochemical and behavioral effects of chronic neuroinflammation in young rats produced by injection of lipopolysaccharide ŽLPS. into the 4th ventricle. The 37-day infusion of LPS impaired spatial memory but not object recognition ability. Electron microscopic studies of neurons within the hippocampus identified numerous paired cisternae of the rough endoplasmic reticulum ŽRER. and other ultrastructural changes that suggested impaired or reduced synthesis of cellular proteins within the cytoplasm. Immunohistochemical staining found numerous highly activated microglia distributed throughout the cingulate gyrus, entorhinal cortex, hippocampus and dentate gyrus. This animal model may be useful to test potential pharmacotherapies that are directed at the prevention of the cytotoxic consequences of chronic neuroinflammation associated with normal aging or Alzheimer’s disease. q 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Rat; Neuroinflammation; Spatial memory; Rough endoplasmic reticulum; Ultrastructural analysis
1. Introduction
2. Experimental procedures
Lipopolysaccharide ŽLPS. is a component of the cell wall of gram-negative bacteria and has been used experimentally to stimulate production of endogenous cytokines and complement proteins w1,17x. Recently, LPS was used to study the cytotoxic effects of chronically elevated cytokine levels within the brain of young rats w9,17,21,22x. Activated glia are the main source of the interleukins ŽIL., IL-1, IL-6 and IL-8, tumor necrosis factor-a ŽTNFa . and prostanoids after an injection of LPS into the brain w1,8,15,17,23x. Activated microglia and elevated cytokine levels are also associated with neuropathological insults and neurodegenerative disorders, such as Alzheimer’s disease w7x. The current study investigated the behavioral and ultrastructural effects of chronic neuroinflammation produced by infusion of LPS into the brains of young rats.
2.1. Subjects
) Corresponding author. [email protected]
Fax: q1-602-626-2618;
e-m ail:
Twenty-eight male Sprague–Dawley rats, approximately 6 months old and weighing 300 g, were housed in a colony room with a 12:12 dark:light cycle with lights off at 1000 h. Behavioral testing was conducted during the dark portion of the cycle. 2.2. Surgical procedures Standard procedures were used for the surgery w9x. Each rat was first given 0.3 ml of atropine methylbromide Ž5 mgrml, i.p.. and then anesthetized with pentobarbital Ž50 mgrkg, i.p... The rat was placed in a stereotaxic instrument with the incisor bar set 3 mm below the earbars Ži.e., flat skull., and the scalp was incised and retracted. A hole was drilled in appropriate location in the skull with a dental drill. Coordinates for the 4th ventricle infusions were as follows: AP: y2.5 mm to Lambda, on the mid-
0006-8993r00r$ - see front matter q 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 0 0 . 0 1 9 9 9 - 5
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Fig. 1. Semi-thin sections of rat hippocampus ŽCornu Ammonis. stained with toluidine blue. ŽA. Control. Most of the neurons are intensely stained and pale semilunar areas presumably corresponding to the Golgi apparatus are often seen in the paranuclear region Žarrow heads.. ŽB. LPS-treated. The perikaryon of all the neurons is faintly stained. In the nucleus, the nucleolus is often located in an eccentric position Žarrows.. Original magnification, 800 = ; bar s 12.5 mm. BV s blood vessel.
line, and DV: y7.0 mm from the dura. An Alzet ŽPalo Alto, CA. osmotic minipump Žmodel 2004; 0.25 mlrh. containing LPS ŽSigma; Escherichia coli, serotype 055:B5, TCA extraction; 1.0 mgrml. was implanted into the dorsal abdomen, between the skin and abdominal wall, and attached via Tygon tubing Ž0.060Y o.d.. to a chronic indwelling cannula ŽModel 3280P, Osmotic pump connect, 28 gauge, Plastics One, Roanoke, VA. that had been positioned stereotaxically so that the cannula tip extended to the coordinates outlined above. Sixteen rats were prepared for LPS infusion; 10 of these rats underwent behavioral testing and six were used for the ultrastructural
analyses. Twelve rats served as controls and were infused with artificial CSF ŽCSF.: Žin mM. 140 NaCl; 3.0 KCl; 2.5 CaCl 2 ; 1.0 MgCl 2 ; 1.2 Na 2 HPO4 , pH 7.4. Ten of these control rats underwent behavioral testing while two were used for the ultrastructural analyses. A volume overload to the CSF space was discounted because the 0.25 mlrh administered contributed to only about 0.2% of the CSF volume produced by the rat each hour and was only 0.15% of the rat’s total CSF volume. In order to determine whether chronic administration of LPS into the 4th ventricle induced a peripheral inflammatory response, TNFa levels were assayed by ELISA ŽNEN, Boston, MA. in
Fig. 2. Electron micrographs of rat hippocampal neurons. ŽA. Control. The neuron has numerous polyribosomes, a well-developed RER and an extended Golgi apparatus. ŽB. LPS-treated. The perikaryon is poor in polyribosomes and RER cisternae which are often paired between themselves andror with the nuclear cisterna; in correspondence of these areas, small, dense masses of heterochromatin were closely apposed to the inner nuclear envelope Žsmall arrows.. Golgi apparatus is poorly developed. The nuclear envelope forms a deep invagination ŽU . containing cytoplasm rich in polyribosomes. Note the presence of myelinic figures within the nucleus Žlarge arrows.. Original magnification, 7500 = , bar s 1.3 mm. N s nucleus, G s Golgi complex.
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samples of serum obtained from each rat Žtail vein bleed. on day 25 of the infusion. Serum samples were also collected and assayed for TNFa at the time of sacrifice. Post-operative care included chloramphenicol Ž1% solution. applied to the exposed skull and scalp prior to closure, lidocaine applied locally to the scalp to lessen pain, and 5 ml of sterile isotonic saline injected Žsubcutaneously. to prevent dehydration during recovery. The rats were closely monitored during recovery and kept under a heat lamp until they were awake and active. Body weights were regularly monitored, and nutritional supplements were provided as necessary during the infusion period. 2.3. BehaÕioral testing Behavioral testing began for 20 rats Ž10 controls and 10 LPS-infused. 10 days after the minipumps had completely expelled their contents Žaccording to information provided by the manufacturer.. This was done in order to allow the acute soporific effects of the LPS infusion to dissipate w12x. The rats were tested on two different behavioral tasks in a counter-balanced order with 5 days separating testing in each task: the T-maze spontaneous alternation task as a test of spatial working memory and an object recognition task. The T-maze task was chosen because accurate performance can be impaired by injury within the hippocampus w16x and because our previous studies have found evidence for extensive inflammation associated with hippocampal pyramidal cell loss w9x. Performance in the object recognition task is not impaired by hippocampal injury w18x. 2.4. Spontaneous alternation performance in a T-maze Each rat was initially allowed to spend 6 min exploring the maze on the day before testing began. For each trial, the rat was placed at the starting point at the base of the stem of the maze and allowed to walk down the stem and choose freely either the left or right arm of the T-maze. The rat was then removed from the maze and placed into a holding cage near the maze. Thirty seconds later, the rat was placed back at the starting point and allowed to walk down the stem and choose either arm. This process was repeated six times in a day. Three and 6 days later, this procedure was repeated. The percent alternation performance each day was averaged across all 3 days of testing; these data were analyzed by Student’s t-test. 2.5. Object recognition task On the first day of testing, each rat was placed in a small open area containing two novel objects. The objects were children’s toys that were all of similar size, surface complexity, and material. The rat was allowed to explore the open field and objects for 6 min and then removed and placed back into its home cage. On the next day, the rat
was placed into the same open field that now contained two objects: one familiar from the previous day and a novel object. The time that each rat spent investigating each object was measured. After 4 min, the rat was removed. The particular novel object presented to each rat was randomized among eight different objects. On the next day, the rat was returned to the open field that now contained a familiar object from the previous day of testing Žrandomized to include either the original familiar or novel object from the previous day. plus a new novel object. The time that each rat spent investigating each object was determined. This procedure was repeated the next day. The percent time spent investigating the novel object on all three of the test days was determined by dividing the absolute time spent investigating the novel object by the total time spent investigating both objects.
2.6. Processing of brain tissue Following behavioral testing, and approximately 70 days after surgery, the rats were killed by a lethal injection of pentobarbital followed by transcardial perfusion of the brain with cold saline containing heparin, prior to being perfused with Žfiltered. 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.3. The brains were removed, post-fixed in 4% paraformaldehyder0.1 M sodium phosphate buffer for 1 h, cryoprotected in 0.1 M sodium phosphate buffer containing 20% sucrose at 48C for 24 h, snap-frozen by transfer into isopentane Žy508C. and stored Žy708C.. Each frozen brain was cut in the coronal plane Ž40 mm. on a cryostat for histological analysis. For electron microscopy, rats were decapitated and their brains were removed, sectioned into 2 mm slabs and than placed in a fixative solution of 2% sodium cacodylatebuffered glutaraldehyde, pH 7.4, for 6 h. The tissues were then rinsed in a buffered solution of saccharose, post-fixed with 1% phosphate-buffered OsO4 , pH 7.4, dehydrated with acetone and embedded in Epon, using flat molds. The semi-thin sections, obtained with an ultramicrotome, were stained with a solution of toluidine blue and photographed under a light microscope ŽLM.. Ultra-thin sections of selected areas were obtained using a diamond knife and stained with an alcoholic solution of uranyl acetate, followed by a solution of concentrated bismuth subnitrate. These sections were examined and photographed under a JEOL 1010 electron microscope ŽEM.. Activated microglia rapidly upregulate their expression of tissue antigens following inflammation w5x. To visualize activated microglia, a monoclonal antibody ŽOX-6, 1r400 dilution, PharMingen, San Diego, CA. directed against the Class II major histocompatibility complex ŽMHC II. was used w6x. Activated astrocytes were visualized using a polyclonal antibody against GFAP Ž1r100 dilution, Sigma..
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Fig. 3. Electron micrographs of rat hippocampal neurons. ŽA. Control. Note the richness in polyribosomes and long RER cisternae. The arrows indicate paired RER cisternae. The Golgi apparatus is prominent and shows signs of intense activity ŽU indicates a Golgi fenestrated cisterna.. ŽB. LPS-treated. Note the limited number of polyribosomes and RER cisternae, most of which are short and paired both between themselves or with the nuclear cisterna Žarrows.. Note the presence of three long cisternae paired for long tracts. The arrowhead indicates three paired RER cisternae. Original magnification, 20,000 = . Bar s 0.5 mm.
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Fig. 4. Electron micrographs of the hippocampal neurons of the LPS-treated rats. ŽA. Nucleus with a ‘‘cerebriform’’ aspect because of the numerous and branched invaginations of the nuclear envelope ŽU .. Original magnification, 15,000 = . Bar s 0.7 mm. ŽB. Numerous pairs of RER cisternae Žarrows.. One of them is paired with the nuclear cisterna. Original magnification, 20,000 = . ŽC. Numerous myelinic figures are contained in the nucleus Žarrowheads. and one in a nuclear envelope evagination ŽU .. Original magnification, 20,000 = . ŽD. Several RER cisternae are paired with the nuclear cisterna and at these levels small and dense masses of heterochromatin are closely apposed to the inner nuclear envelope Žarrows.. U indicates a nuclear envelope invagination. Original magnification, 20,000 = . Bar s 0.5 mm, G s Golgi complex.
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3. Results The chronic infusion of LPS did not induce seizures or fever and was well tolerated by all of the rats. Initially after surgery, all of the LPS-treated rats lost a few grams of weight. Within a few days, however, all of these rats had regained weight and continued to grow normally during and after the infusion period. Serum levels of TNFa on day 25, or on the day of sacrifice, were not elevated by the LPS infusion. 3.1. BehaÕior Chronic infusion of LPS into the 4th ventricle for 37 days significantly Ž t s 6.58, df s 18, p - 0.001. impaired alternation performance ŽLPS, 35.4 " 3.4 vs. Control, 80.1 " 6.55, mean " S.E.M... The chronic LPS infusion had no effect upon object recognition performance Ž t s 0.24, df s 18, p s 0.81.. 3.2. Histology The possible effects of LPS treatment on the structure and ultrastructure of hippocampal neurons ŽCornu Ammonis, CA1. and glia were investigated in semi- and ultra-thin sections by using LM and EM, respectively. Under LM examination of the semi-thin sections in the CSF-treated rats, most of the hippocampal neurons were intensely
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stained and often had pale semilunar areas, corresponding to the Golgi apparatus, in the perinuclear region ŽFig. 1A.. In contrast, hippocampal neurons in the LPS-treated rats showed a pale cytoplasm and the nucleus often had an irregular contour and an eccentric nucleolus ŽFig. 1B.. 3.2.1. EM changes in cytoplasm In CSF-treated rats, the cytoplasm of the hippocampal neurons was filled with polyribosomes and rough endoplasmic reticulum ŽRER. cisternae ŽFig. 2A.. The latter were long and sometimes paired to each other for short tracts. Along the paired tracts, the facing membranes were devoid of ribosomes and the interposed cytoplasm was moderately electron-dense ŽFig. 3A.. The Golgi complexes were numerous and prominent ŽFig. 2A, Fig. 3A.. In the LPS-treated rats, on the contrary, the cytoplasm of the hippocampal neurons had an empty aspect and contained much fewer polyribosomes and RER cisternae ŽFig. 2B.. These cisternae were very often paired ŽFig. 3B, Fig. 4B.. The Golgi complexes were always small ŽFig. 2B, Fig. 4B,C,D.. Overall, the appearance of the mitochondria, lysosomes, lipofuscin pigment and cytoskeleton apparatus did not differ between LPS- and CSF-treated rats. 3.2.2. EM changes within the nucleus In CSF-treated rats, the nucleus had a round shape and regular or slightly undulated contours ŽFig. 2A, Fig. 3A.. In contrast, in the LPS-treated rats, the nucleus showed an
Fig. 5. Electron micrograph of a hippocampal intraparenchymal microglial cell of LPS-treated rat. The Golgi apparatus ŽG. is very extended and the RER cisternae and mitochondria are numerous. Original magnification, 12500= . Bar s 0.8 mm. ŽInset. Semi-thin section. Activated microglia cell showing dark cytoplasm. Original magnification, 800 = . Bar s 12.5 mm.
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Fig. 6. Immunohistochemical staining for the MHC II Žusing the OX6 antibody.. ŽA. Controls. A few OX6-positive microglia were seen in the brains of aCSF-treated rats. ŽB. LPS-treated. Activated microglia distributed throughout the entorhinal cortex and hippocampus; the highest concentration was within the dentate gyrus. The reactive microglia in the LPS-treated rats were characterized by a contraction of their highly ramified processes that appeared bushy in morphology. Original magnification, 110 = .
extremely irregular contour due to the presence of deep, tubular invaginations of the nuclear envelope containing wide areas of cytoplasm that were particularly rich in polyribosomes ŽFig. 2B, Fig. 4A,C,D.. In some neurons of LPS-treated rats, these invaginations were numerous and branched, giving to the nucleus a ‘‘cerebriform’’ aspect ŽFig. 4A.. Sections of the nuclear envelope were often paired with one cisterna ŽFig. 3B, Fig. 4. or with a pair of cisternae of the RER ŽFig. 4B.; often, in the correspondence of these areas, small, dense masses of heterochromatin were closely apposed to the inner nuclear membrane ŽFig. 2B, Fig. 4D.. Myelinic figures were seen in the nuclear matrix ŽFig. 2B, Fig. 4C. and in wide evaginations of the perinuclear cisterna ŽFig. 4C.. The LM and EM investigations demonstrated that in the LPS-treated rats the intraparenchymal Žperineuronal. microglia were intensely activated. This activated microglia cell had a large and oval-shaped body and a dark cytoplasm. Žinset in Fig. 5.. Under EM examination, the cytoplasm of these activated microglia had a highly extended RER, an enormous Golgi apparatus with large cisternae and numerous mitochondria ŽFig. 5.. These changes are typical of activated microglia associated with inflammation in the brain w11x. Following chronic exposure to LPS, immunohistochemical staining for the MHC II Žusing the OX6 antibody.
found numerous, highly activated microglia distributed throughout the cingulate gyrus and entorhinal cortex, as well as the hippocampus and dentate gyrus ŽFig. 6B., as compared to the hippocampus and dentate gyrus of CSFtreated rats ŽFig. 6A.. The reactive microglia were characterized by a contraction of their highly ramified processes that appeared bushy in morphology. We also found that the LPS treatment significantly increased the number of GFAP-positive astrocytes throughout the brain Žnot shown., as we have previously shown w9,22x.
4. Discussion The current study confirms and significantly extends the results of our earlier studies on the effects of chronic inflammation within the brains of young rats w9,21,23x. Previous studies have demonstrated that the central inflammatory response produced by infusion of LPS into the 4th ventricle is associated with an increase in the number of activated astrocytes and reactive microglia distributed throughout the brain w9,21x; that the greatest inflammatory response occurred within the cingulate gyrus and temporal lobe, particularly the hippocampus, as well as within the basal forebrain w9,21x; and that the inflammation was associated with an induction in IL-1a , IL-1b, and TNFa
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mRNA levels, particularly within the basal forebrain and hippocampus w9x. Finally, we have previously shown that chronic neuroinflammation was associated with the nonapoptotic degeneration of hippocampal pyramidal neurons w9x. In the present study, the neurobiological consequences of the chronic neuroinflammation produced a significant deficit in spatial memory. In contrast, object recognition ability was unaffected. This temporal lobe-dependent behavioral impairment may be due to the degeneration of hippocampal pyramidal neurons induced by the chronic neuroinflammation w9x. Consistent with our current results is a recent report that infusion of IL-2 into a lateral ventricle for only 2 weeks impaired performance in the Morris water maze w8x. This pattern of behavioral results is consistent with that typically seen following experimental hippocampal injury w4,18x. The absence of detectable levels of TNFa in the serum of LPS-treated rats indicates that the inflammation was confined to the central nervous system. Although LPS is a lipophilic molecule, it does not appear to leave the brain in significant amounts when infused centrally w3x. Therefore, a general malaise did not cause the impaired behavioral performance. It is also conceivable that the pathological effects of the chronic LPS infusion are due to non-specific cytotoxic effects of LPS upon the brain. This is highly unlikely inasmuch as we have previously shown that the effects of LPS are selective for cholinergic basal forebrain cells w22x and that the cytotoxicity of LPS can be completely attenuated by co-administration of either cyclooxygenase inhibitors or glutamate receptor antagonists w21x. These findings indicate that the cytotoxic effects of chronic LPS infusion are due to the formation of prostaglandins and the release of endogenous glutamate. The electron microscopic study revealed numerous changes in the cellular components involved in protein synthesis within the hippocampal neurons of LPS-treated rats. These changes probably underlie the significant learning and memory impairments that we have previously described in similarly prepared rats w9x. The cytoplasm of these cells contained only a few polyribosomes, a few cisternae of RER and a small Golgi apparatus, all suggestive of either impaired or reduced protein synthesis. In addition, the presence of myelinic figures within the nucleus and the high concentration of ribosomes sequestered within the nuclear infoldings are consistent with the nuclear dysfunction. However, the presence of deep invaginations of the nuclear envelope would result in an increase in the exchange area between the nuclear matrix and the cytoplasm. The eccentric position of the nucleolus usually occurs in cells actively engaged in protein synthesis. These two ultrastructural changes would represent a possible attempt by the neurons to compensate for impaired protein synthesis within the cytoplasm. Because this study only examined for changes at a single time point, it is not possible to know whether the observed changes reflect
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either progressing or recovering changes in neuronal integrity. The most interesting and unusual electron microscopic finding was that almost all of the RER cisternae were paired between themselves and also with the perinuclear cisterna. The meaning of this pairing is unknown but certainly at the regions of the paired tracts the ribosomes would be prevented from interacting with the RER surface; this would be consistent with a further impairing in protein synthesis. These data are consistent with the hypothesis that the impaired behavioral performance observed might be due to impaired protein synthesis within hippocampal neurons leading to a general functional impairment. In contrast to these changes seen in the nucleus, no problems were associated with the organelles of cellular respiration Žmitochondria. or catalytic processes Žlysosomes, pigments.. The cytoskeleton also appeared normal; these findings would be consistent with normal axonal transport. Furthermore, the present results demonstrated a consistent increase in the number of activated microglia cells in the hippocampus of LPS-treated rats; the ultrastructural investigation confirmed that these cells had the features of intense activation w2x. Recent studies have reported a significant increase in the number of activated microglia and astrocytes within brains of aged rats and non-human primates w7,19,20x. Our data are consistent with the hypothesis that long-term inflammation within the brain may lead to degenerative changes, particularly within the hippocampus. These degenerative changes might underlie the age-associated deficit in spatial memory ability that has been widely documented in rats w14x. The greatly enhanced inflammatory response observed within the temporal lobe and cingulate gyrus in the present and previous studies w9,21x might explain why these brain regions are associated with such profound pathological changes in the brains of patients with Alzheimer’s disease w10,13x.
Acknowledgements Supported by the US Public Health Service, Contract grant number AG10546 and the Alzheimer’s Association, IIRG-95-004, to GLW. We thank Prof. Maria-Simonetta Faussone-Pellegrini for fundamental theoretical support of the electron microscopy, D. Guasti, L. Calosi, C. Righini and P. Guasti for technical assistance with the electron microscopy studies.
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w13x D.M.A. Mann, Pathological correlates of dementia in Alzheimer’s disease, Neurobiol. Aging 15 Ž1994. 357–360. w14x A.L. Markowska, W.S. Stone, D.K. Ingram, J. Reynolds, P.E. Gold, L.H. Conti, M.J. Pontecorvo, G.L. Wenk, D.S. Olton, Individual differences in aging: behavioral and neurobiological correlates, Neurobiol. Aging 10 Ž1989. 31–43. w15x L. Minghetti, G. Levi, Induction of prostanoid biosynthesis by bacterial lipopolysaccharide and isoproterenol in rat microglial cultures, J. Neurochem. 65 Ž1995. 2690–2698. w16x D.S. Olton, B.C. Papas, Spatial memory and hippocampal function, Neuropsychology 17 Ž1979. 669–682. w17x N. Quan, S.K. Sundar, J.M. Weiss, Induction of interleukin-1 in various brain regions after peripheral and central injections of lipopolysaccharide, J. Neuroimmunol. 49 Ž1994. 125–134. w18x L.A. Rothblat, L.F. Kromer, Object recognition memory in the rat: the role of the hippocampus, Behav. Brain Res. 42 Ž1991. 25–32. w19x I. Rozovsky, C.E. Finch, T.E. Morgan, Age-related activation of microglia and astrocytes — in vitro studies show persistent phenotypes of aging, increased proliferation, and resistance to down-regulation, Neurobiol. Aging 19 Ž1998. 97–103. w20x L.G. Sheffield, N.E.J. Berman, Microglial expression of MHC class II increases in normal aging of nonhuman primates, Neurobiol. Aging 19 Ž1998. 47–55. w21x G.L. Wenk, B. Hauss-Wegrzyniak, L.B. Willard, Pathological and biochemical studies of chronic neuroinflammation may lead to therapies for Alzheimer’s disease, in: P. Patterson, C. Kordon, Y. Christen ŽEds.., Research and Perspectives in Neurosciences: Neuro -Immune Neurodegenerative and Psychiatric Disorders and Neural Injury, Springer-Verlag, Heidelberg, 1999, pp. 73–77. w22x L.B. Willard, B. Hauss-Wegrzyniak, G.L. Wenk, The pathological and biochemical consequences of acute and chronic neuroinflammation within the basal forebrain of rats, Neuroscience 88 Ž1999. 193–200. w23x J. Yao, R.W. Johnson, Induction of interleukin-1b-converting enzyme ŽICE. in murine microglia by lipopolysaccharide, Mol. Brain Res. 51 Ž1997. 170–178.
Research report
Behavioral and ultrastructural changes induced by chronic neuroinflammation in young rats Beatrice Hauss-Wegrzyniak a , Maria G. Vannucchi b , Gary L. Wenk a
a, )
Arizona Research Laboratories, DiÕision of Neural Systems, Memory and Aging, 350 Life Sciences North Building, UniÕersity of Arizona, Tucson, AZ 85724, USA b Department of Anatomy and Histology, UniÕersity of Florence, Florence, Italy Accepted 4 January 2000
Abstract We investigated the ultrastructural, immunohistochemical, biochemical and behavioral effects of chronic neuroinflammation in young rats produced by injection of lipopolysaccharide ŽLPS. into the 4th ventricle. The 37-day infusion of LPS impaired spatial memory but not object recognition ability. Electron microscopic studies of neurons within the hippocampus identified numerous paired cisternae of the rough endoplasmic reticulum ŽRER. and other ultrastructural changes that suggested impaired or reduced synthesis of cellular proteins within the cytoplasm. Immunohistochemical staining found numerous highly activated microglia distributed throughout the cingulate gyrus, entorhinal cortex, hippocampus and dentate gyrus. This animal model may be useful to test potential pharmacotherapies that are directed at the prevention of the cytotoxic consequences of chronic neuroinflammation associated with normal aging or Alzheimer’s disease. q 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Rat; Neuroinflammation; Spatial memory; Rough endoplasmic reticulum; Ultrastructural analysis
1. Introduction
2. Experimental procedures
Lipopolysaccharide ŽLPS. is a component of the cell wall of gram-negative bacteria and has been used experimentally to stimulate production of endogenous cytokines and complement proteins w1,17x. Recently, LPS was used to study the cytotoxic effects of chronically elevated cytokine levels within the brain of young rats w9,17,21,22x. Activated glia are the main source of the interleukins ŽIL., IL-1, IL-6 and IL-8, tumor necrosis factor-a ŽTNFa . and prostanoids after an injection of LPS into the brain w1,8,15,17,23x. Activated microglia and elevated cytokine levels are also associated with neuropathological insults and neurodegenerative disorders, such as Alzheimer’s disease w7x. The current study investigated the behavioral and ultrastructural effects of chronic neuroinflammation produced by infusion of LPS into the brains of young rats.
2.1. Subjects
) Corresponding author. [email protected]
Fax: q1-602-626-2618;
e-m ail:
Twenty-eight male Sprague–Dawley rats, approximately 6 months old and weighing 300 g, were housed in a colony room with a 12:12 dark:light cycle with lights off at 1000 h. Behavioral testing was conducted during the dark portion of the cycle. 2.2. Surgical procedures Standard procedures were used for the surgery w9x. Each rat was first given 0.3 ml of atropine methylbromide Ž5 mgrml, i.p.. and then anesthetized with pentobarbital Ž50 mgrkg, i.p... The rat was placed in a stereotaxic instrument with the incisor bar set 3 mm below the earbars Ži.e., flat skull., and the scalp was incised and retracted. A hole was drilled in appropriate location in the skull with a dental drill. Coordinates for the 4th ventricle infusions were as follows: AP: y2.5 mm to Lambda, on the mid-
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Fig. 1. Semi-thin sections of rat hippocampus ŽCornu Ammonis. stained with toluidine blue. ŽA. Control. Most of the neurons are intensely stained and pale semilunar areas presumably corresponding to the Golgi apparatus are often seen in the paranuclear region Žarrow heads.. ŽB. LPS-treated. The perikaryon of all the neurons is faintly stained. In the nucleus, the nucleolus is often located in an eccentric position Žarrows.. Original magnification, 800 = ; bar s 12.5 mm. BV s blood vessel.
line, and DV: y7.0 mm from the dura. An Alzet ŽPalo Alto, CA. osmotic minipump Žmodel 2004; 0.25 mlrh. containing LPS ŽSigma; Escherichia coli, serotype 055:B5, TCA extraction; 1.0 mgrml. was implanted into the dorsal abdomen, between the skin and abdominal wall, and attached via Tygon tubing Ž0.060Y o.d.. to a chronic indwelling cannula ŽModel 3280P, Osmotic pump connect, 28 gauge, Plastics One, Roanoke, VA. that had been positioned stereotaxically so that the cannula tip extended to the coordinates outlined above. Sixteen rats were prepared for LPS infusion; 10 of these rats underwent behavioral testing and six were used for the ultrastructural
analyses. Twelve rats served as controls and were infused with artificial CSF ŽCSF.: Žin mM. 140 NaCl; 3.0 KCl; 2.5 CaCl 2 ; 1.0 MgCl 2 ; 1.2 Na 2 HPO4 , pH 7.4. Ten of these control rats underwent behavioral testing while two were used for the ultrastructural analyses. A volume overload to the CSF space was discounted because the 0.25 mlrh administered contributed to only about 0.2% of the CSF volume produced by the rat each hour and was only 0.15% of the rat’s total CSF volume. In order to determine whether chronic administration of LPS into the 4th ventricle induced a peripheral inflammatory response, TNFa levels were assayed by ELISA ŽNEN, Boston, MA. in
Fig. 2. Electron micrographs of rat hippocampal neurons. ŽA. Control. The neuron has numerous polyribosomes, a well-developed RER and an extended Golgi apparatus. ŽB. LPS-treated. The perikaryon is poor in polyribosomes and RER cisternae which are often paired between themselves andror with the nuclear cisterna; in correspondence of these areas, small, dense masses of heterochromatin were closely apposed to the inner nuclear envelope Žsmall arrows.. Golgi apparatus is poorly developed. The nuclear envelope forms a deep invagination ŽU . containing cytoplasm rich in polyribosomes. Note the presence of myelinic figures within the nucleus Žlarge arrows.. Original magnification, 7500 = , bar s 1.3 mm. N s nucleus, G s Golgi complex.
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samples of serum obtained from each rat Žtail vein bleed. on day 25 of the infusion. Serum samples were also collected and assayed for TNFa at the time of sacrifice. Post-operative care included chloramphenicol Ž1% solution. applied to the exposed skull and scalp prior to closure, lidocaine applied locally to the scalp to lessen pain, and 5 ml of sterile isotonic saline injected Žsubcutaneously. to prevent dehydration during recovery. The rats were closely monitored during recovery and kept under a heat lamp until they were awake and active. Body weights were regularly monitored, and nutritional supplements were provided as necessary during the infusion period. 2.3. BehaÕioral testing Behavioral testing began for 20 rats Ž10 controls and 10 LPS-infused. 10 days after the minipumps had completely expelled their contents Žaccording to information provided by the manufacturer.. This was done in order to allow the acute soporific effects of the LPS infusion to dissipate w12x. The rats were tested on two different behavioral tasks in a counter-balanced order with 5 days separating testing in each task: the T-maze spontaneous alternation task as a test of spatial working memory and an object recognition task. The T-maze task was chosen because accurate performance can be impaired by injury within the hippocampus w16x and because our previous studies have found evidence for extensive inflammation associated with hippocampal pyramidal cell loss w9x. Performance in the object recognition task is not impaired by hippocampal injury w18x. 2.4. Spontaneous alternation performance in a T-maze Each rat was initially allowed to spend 6 min exploring the maze on the day before testing began. For each trial, the rat was placed at the starting point at the base of the stem of the maze and allowed to walk down the stem and choose freely either the left or right arm of the T-maze. The rat was then removed from the maze and placed into a holding cage near the maze. Thirty seconds later, the rat was placed back at the starting point and allowed to walk down the stem and choose either arm. This process was repeated six times in a day. Three and 6 days later, this procedure was repeated. The percent alternation performance each day was averaged across all 3 days of testing; these data were analyzed by Student’s t-test. 2.5. Object recognition task On the first day of testing, each rat was placed in a small open area containing two novel objects. The objects were children’s toys that were all of similar size, surface complexity, and material. The rat was allowed to explore the open field and objects for 6 min and then removed and placed back into its home cage. On the next day, the rat
was placed into the same open field that now contained two objects: one familiar from the previous day and a novel object. The time that each rat spent investigating each object was measured. After 4 min, the rat was removed. The particular novel object presented to each rat was randomized among eight different objects. On the next day, the rat was returned to the open field that now contained a familiar object from the previous day of testing Žrandomized to include either the original familiar or novel object from the previous day. plus a new novel object. The time that each rat spent investigating each object was determined. This procedure was repeated the next day. The percent time spent investigating the novel object on all three of the test days was determined by dividing the absolute time spent investigating the novel object by the total time spent investigating both objects.
2.6. Processing of brain tissue Following behavioral testing, and approximately 70 days after surgery, the rats were killed by a lethal injection of pentobarbital followed by transcardial perfusion of the brain with cold saline containing heparin, prior to being perfused with Žfiltered. 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.3. The brains were removed, post-fixed in 4% paraformaldehyder0.1 M sodium phosphate buffer for 1 h, cryoprotected in 0.1 M sodium phosphate buffer containing 20% sucrose at 48C for 24 h, snap-frozen by transfer into isopentane Žy508C. and stored Žy708C.. Each frozen brain was cut in the coronal plane Ž40 mm. on a cryostat for histological analysis. For electron microscopy, rats were decapitated and their brains were removed, sectioned into 2 mm slabs and than placed in a fixative solution of 2% sodium cacodylatebuffered glutaraldehyde, pH 7.4, for 6 h. The tissues were then rinsed in a buffered solution of saccharose, post-fixed with 1% phosphate-buffered OsO4 , pH 7.4, dehydrated with acetone and embedded in Epon, using flat molds. The semi-thin sections, obtained with an ultramicrotome, were stained with a solution of toluidine blue and photographed under a light microscope ŽLM.. Ultra-thin sections of selected areas were obtained using a diamond knife and stained with an alcoholic solution of uranyl acetate, followed by a solution of concentrated bismuth subnitrate. These sections were examined and photographed under a JEOL 1010 electron microscope ŽEM.. Activated microglia rapidly upregulate their expression of tissue antigens following inflammation w5x. To visualize activated microglia, a monoclonal antibody ŽOX-6, 1r400 dilution, PharMingen, San Diego, CA. directed against the Class II major histocompatibility complex ŽMHC II. was used w6x. Activated astrocytes were visualized using a polyclonal antibody against GFAP Ž1r100 dilution, Sigma..
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Fig. 3. Electron micrographs of rat hippocampal neurons. ŽA. Control. Note the richness in polyribosomes and long RER cisternae. The arrows indicate paired RER cisternae. The Golgi apparatus is prominent and shows signs of intense activity ŽU indicates a Golgi fenestrated cisterna.. ŽB. LPS-treated. Note the limited number of polyribosomes and RER cisternae, most of which are short and paired both between themselves or with the nuclear cisterna Žarrows.. Note the presence of three long cisternae paired for long tracts. The arrowhead indicates three paired RER cisternae. Original magnification, 20,000 = . Bar s 0.5 mm.
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Fig. 4. Electron micrographs of the hippocampal neurons of the LPS-treated rats. ŽA. Nucleus with a ‘‘cerebriform’’ aspect because of the numerous and branched invaginations of the nuclear envelope ŽU .. Original magnification, 15,000 = . Bar s 0.7 mm. ŽB. Numerous pairs of RER cisternae Žarrows.. One of them is paired with the nuclear cisterna. Original magnification, 20,000 = . ŽC. Numerous myelinic figures are contained in the nucleus Žarrowheads. and one in a nuclear envelope evagination ŽU .. Original magnification, 20,000 = . ŽD. Several RER cisternae are paired with the nuclear cisterna and at these levels small and dense masses of heterochromatin are closely apposed to the inner nuclear envelope Žarrows.. U indicates a nuclear envelope invagination. Original magnification, 20,000 = . Bar s 0.5 mm, G s Golgi complex.
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3. Results The chronic infusion of LPS did not induce seizures or fever and was well tolerated by all of the rats. Initially after surgery, all of the LPS-treated rats lost a few grams of weight. Within a few days, however, all of these rats had regained weight and continued to grow normally during and after the infusion period. Serum levels of TNFa on day 25, or on the day of sacrifice, were not elevated by the LPS infusion. 3.1. BehaÕior Chronic infusion of LPS into the 4th ventricle for 37 days significantly Ž t s 6.58, df s 18, p - 0.001. impaired alternation performance ŽLPS, 35.4 " 3.4 vs. Control, 80.1 " 6.55, mean " S.E.M... The chronic LPS infusion had no effect upon object recognition performance Ž t s 0.24, df s 18, p s 0.81.. 3.2. Histology The possible effects of LPS treatment on the structure and ultrastructure of hippocampal neurons ŽCornu Ammonis, CA1. and glia were investigated in semi- and ultra-thin sections by using LM and EM, respectively. Under LM examination of the semi-thin sections in the CSF-treated rats, most of the hippocampal neurons were intensely
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stained and often had pale semilunar areas, corresponding to the Golgi apparatus, in the perinuclear region ŽFig. 1A.. In contrast, hippocampal neurons in the LPS-treated rats showed a pale cytoplasm and the nucleus often had an irregular contour and an eccentric nucleolus ŽFig. 1B.. 3.2.1. EM changes in cytoplasm In CSF-treated rats, the cytoplasm of the hippocampal neurons was filled with polyribosomes and rough endoplasmic reticulum ŽRER. cisternae ŽFig. 2A.. The latter were long and sometimes paired to each other for short tracts. Along the paired tracts, the facing membranes were devoid of ribosomes and the interposed cytoplasm was moderately electron-dense ŽFig. 3A.. The Golgi complexes were numerous and prominent ŽFig. 2A, Fig. 3A.. In the LPS-treated rats, on the contrary, the cytoplasm of the hippocampal neurons had an empty aspect and contained much fewer polyribosomes and RER cisternae ŽFig. 2B.. These cisternae were very often paired ŽFig. 3B, Fig. 4B.. The Golgi complexes were always small ŽFig. 2B, Fig. 4B,C,D.. Overall, the appearance of the mitochondria, lysosomes, lipofuscin pigment and cytoskeleton apparatus did not differ between LPS- and CSF-treated rats. 3.2.2. EM changes within the nucleus In CSF-treated rats, the nucleus had a round shape and regular or slightly undulated contours ŽFig. 2A, Fig. 3A.. In contrast, in the LPS-treated rats, the nucleus showed an
Fig. 5. Electron micrograph of a hippocampal intraparenchymal microglial cell of LPS-treated rat. The Golgi apparatus ŽG. is very extended and the RER cisternae and mitochondria are numerous. Original magnification, 12500= . Bar s 0.8 mm. ŽInset. Semi-thin section. Activated microglia cell showing dark cytoplasm. Original magnification, 800 = . Bar s 12.5 mm.
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Fig. 6. Immunohistochemical staining for the MHC II Žusing the OX6 antibody.. ŽA. Controls. A few OX6-positive microglia were seen in the brains of aCSF-treated rats. ŽB. LPS-treated. Activated microglia distributed throughout the entorhinal cortex and hippocampus; the highest concentration was within the dentate gyrus. The reactive microglia in the LPS-treated rats were characterized by a contraction of their highly ramified processes that appeared bushy in morphology. Original magnification, 110 = .
extremely irregular contour due to the presence of deep, tubular invaginations of the nuclear envelope containing wide areas of cytoplasm that were particularly rich in polyribosomes ŽFig. 2B, Fig. 4A,C,D.. In some neurons of LPS-treated rats, these invaginations were numerous and branched, giving to the nucleus a ‘‘cerebriform’’ aspect ŽFig. 4A.. Sections of the nuclear envelope were often paired with one cisterna ŽFig. 3B, Fig. 4. or with a pair of cisternae of the RER ŽFig. 4B.; often, in the correspondence of these areas, small, dense masses of heterochromatin were closely apposed to the inner nuclear membrane ŽFig. 2B, Fig. 4D.. Myelinic figures were seen in the nuclear matrix ŽFig. 2B, Fig. 4C. and in wide evaginations of the perinuclear cisterna ŽFig. 4C.. The LM and EM investigations demonstrated that in the LPS-treated rats the intraparenchymal Žperineuronal. microglia were intensely activated. This activated microglia cell had a large and oval-shaped body and a dark cytoplasm. Žinset in Fig. 5.. Under EM examination, the cytoplasm of these activated microglia had a highly extended RER, an enormous Golgi apparatus with large cisternae and numerous mitochondria ŽFig. 5.. These changes are typical of activated microglia associated with inflammation in the brain w11x. Following chronic exposure to LPS, immunohistochemical staining for the MHC II Žusing the OX6 antibody.
found numerous, highly activated microglia distributed throughout the cingulate gyrus and entorhinal cortex, as well as the hippocampus and dentate gyrus ŽFig. 6B., as compared to the hippocampus and dentate gyrus of CSFtreated rats ŽFig. 6A.. The reactive microglia were characterized by a contraction of their highly ramified processes that appeared bushy in morphology. We also found that the LPS treatment significantly increased the number of GFAP-positive astrocytes throughout the brain Žnot shown., as we have previously shown w9,22x.
4. Discussion The current study confirms and significantly extends the results of our earlier studies on the effects of chronic inflammation within the brains of young rats w9,21,23x. Previous studies have demonstrated that the central inflammatory response produced by infusion of LPS into the 4th ventricle is associated with an increase in the number of activated astrocytes and reactive microglia distributed throughout the brain w9,21x; that the greatest inflammatory response occurred within the cingulate gyrus and temporal lobe, particularly the hippocampus, as well as within the basal forebrain w9,21x; and that the inflammation was associated with an induction in IL-1a , IL-1b, and TNFa
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mRNA levels, particularly within the basal forebrain and hippocampus w9x. Finally, we have previously shown that chronic neuroinflammation was associated with the nonapoptotic degeneration of hippocampal pyramidal neurons w9x. In the present study, the neurobiological consequences of the chronic neuroinflammation produced a significant deficit in spatial memory. In contrast, object recognition ability was unaffected. This temporal lobe-dependent behavioral impairment may be due to the degeneration of hippocampal pyramidal neurons induced by the chronic neuroinflammation w9x. Consistent with our current results is a recent report that infusion of IL-2 into a lateral ventricle for only 2 weeks impaired performance in the Morris water maze w8x. This pattern of behavioral results is consistent with that typically seen following experimental hippocampal injury w4,18x. The absence of detectable levels of TNFa in the serum of LPS-treated rats indicates that the inflammation was confined to the central nervous system. Although LPS is a lipophilic molecule, it does not appear to leave the brain in significant amounts when infused centrally w3x. Therefore, a general malaise did not cause the impaired behavioral performance. It is also conceivable that the pathological effects of the chronic LPS infusion are due to non-specific cytotoxic effects of LPS upon the brain. This is highly unlikely inasmuch as we have previously shown that the effects of LPS are selective for cholinergic basal forebrain cells w22x and that the cytotoxicity of LPS can be completely attenuated by co-administration of either cyclooxygenase inhibitors or glutamate receptor antagonists w21x. These findings indicate that the cytotoxic effects of chronic LPS infusion are due to the formation of prostaglandins and the release of endogenous glutamate. The electron microscopic study revealed numerous changes in the cellular components involved in protein synthesis within the hippocampal neurons of LPS-treated rats. These changes probably underlie the significant learning and memory impairments that we have previously described in similarly prepared rats w9x. The cytoplasm of these cells contained only a few polyribosomes, a few cisternae of RER and a small Golgi apparatus, all suggestive of either impaired or reduced protein synthesis. In addition, the presence of myelinic figures within the nucleus and the high concentration of ribosomes sequestered within the nuclear infoldings are consistent with the nuclear dysfunction. However, the presence of deep invaginations of the nuclear envelope would result in an increase in the exchange area between the nuclear matrix and the cytoplasm. The eccentric position of the nucleolus usually occurs in cells actively engaged in protein synthesis. These two ultrastructural changes would represent a possible attempt by the neurons to compensate for impaired protein synthesis within the cytoplasm. Because this study only examined for changes at a single time point, it is not possible to know whether the observed changes reflect
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either progressing or recovering changes in neuronal integrity. The most interesting and unusual electron microscopic finding was that almost all of the RER cisternae were paired between themselves and also with the perinuclear cisterna. The meaning of this pairing is unknown but certainly at the regions of the paired tracts the ribosomes would be prevented from interacting with the RER surface; this would be consistent with a further impairing in protein synthesis. These data are consistent with the hypothesis that the impaired behavioral performance observed might be due to impaired protein synthesis within hippocampal neurons leading to a general functional impairment. In contrast to these changes seen in the nucleus, no problems were associated with the organelles of cellular respiration Žmitochondria. or catalytic processes Žlysosomes, pigments.. The cytoskeleton also appeared normal; these findings would be consistent with normal axonal transport. Furthermore, the present results demonstrated a consistent increase in the number of activated microglia cells in the hippocampus of LPS-treated rats; the ultrastructural investigation confirmed that these cells had the features of intense activation w2x. Recent studies have reported a significant increase in the number of activated microglia and astrocytes within brains of aged rats and non-human primates w7,19,20x. Our data are consistent with the hypothesis that long-term inflammation within the brain may lead to degenerative changes, particularly within the hippocampus. These degenerative changes might underlie the age-associated deficit in spatial memory ability that has been widely documented in rats w14x. The greatly enhanced inflammatory response observed within the temporal lobe and cingulate gyrus in the present and previous studies w9,21x might explain why these brain regions are associated with such profound pathological changes in the brains of patients with Alzheimer’s disease w10,13x.
Acknowledgements Supported by the US Public Health Service, Contract grant number AG10546 and the Alzheimer’s Association, IIRG-95-004, to GLW. We thank Prof. Maria-Simonetta Faussone-Pellegrini for fundamental theoretical support of the electron microscopy, D. Guasti, L. Calosi, C. Righini and P. Guasti for technical assistance with the electron microscopy studies.
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