Tampere School of Public Health, FIN-33014 University of Tampere, Tampere,
1 Department of Internal Medicine, Tampere University Hospital, Tampere,
2 Department of Medicine, Division of Nephrology, Helsinki University Central Hospital, Helsinki and
3 Institute of Biomedicine, Helsinki, Finland
Received 20 April 2001; in revised form 19 October 2001; accepted 30 January 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Microglial cells are found throughout the central nervous system (CNS). They are believed to play an active role in CNS inflammatory, immune and degenerative processes, but also in the normal physiology of the brain. Their morphology, immunophenotype and function resemble those of monocytes and macrophages (see Nakajima and Kohsaka, 1993, for a review). Microglia accumulate, proliferate, become activated and phagocytose degenerated neurons in brain ischaemia (Gehrmann et al., 1992
; Lees, 1993
), Alzheimers disease (Perlmutter et al., 1990
) and multiple sclerosis (Banati and Graeber, 1994
). Microglial cells are sensors of brain pathology, and are able to react to disturbances in neuronal well-being.
Kupffer cells, the resident macrophages in the liver, are activated during heavy ethanol administration (Thurman, 1998), and also during ethanol withdrawal (Bautista and Spitzer, 1992
; Lukkari et al., 1999
). It is suggested that this Kupffer cell activation causes hepatic hypoxia and increases free radical production, thereby contributing to ethanol-induced liver injury (Thurman, 1998
). The impact of cerebellar macrophages, i.e. microglia, on alcohol-induced neuronal damage has not been studied so far. The aim of the present study was to ascertain whether ethanol exposure affects the number of cerebellar microglia, and whether the pattern of ethanol exposure (continuous vs intermittent) plays a decisive role in this regard. In particular, we studied folia II and X in the cerebellar vermis. Folium II was selected as previous studies have suggested that it is probably the most vulnerable area to ethanol-induced degeneration (Viktor et al., 1959
; Torvik and Torp, 1986
; Rintala et al., 1997
). Folium X was used as a reference area.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
At the beginning of the experiment, rats were divided into three groups: a control group (n = 6), a continuously ethanol-exposed group (n = 6) and an intermittently ethanol-exposed group (n = 6). The only available fluid throughout the experiment was tap water for the control group and 10% (v/v) ethanol for the continuous group. The intermittent group was given 10% (v/v) ethanol on Monday, Tuesday, Thursday and Friday, and water for the rest of the week. This weekly schedule (two withdrawal periods per week) was repeated throughout the 51/2-month experiment. Food (Ewos R36; Ewos AB, Sweden) was available ad libitum for all groups.
Methods
The rats were decapitated under deep pentobarbital anaesthesia. The brains were quickly removed, and the cerebellum carefully dissected. The vermis was separated from the cerebellar hemispheres, and fixed in 4% paraformaldehyde for 24 h. The vermis was cryoprotected with ascending concentrations of sucrose (10, 20 and 30% sucrose in phosphate-buffered saline). The whole vermis was parasagittally sectioned to the thickness of 10 µm at -15°C. Every 36th section was collected from a random starting point for the morphometric analysis (section sampling fraction = 1/36 and the reciprocal sampling fraction s = 36). The next two sections were also collected for histochemical stainings. The presence of folium X was used to determine the beginning and the end of the vermis, because it has only a rudimentary hemispheric part (Heinsen and Heinsen, 1984). Folium X was also used as a reference area in the morphometric analysis, as the volume measurements were most reliable on folium X, due to its negligible hemispheric extent.
The sections used in the volumetric analysis were stained using 0.05% cresyl violet acetate, dehydrated in ascending ethanol (50, 75, 96 and 100%), cleared with xylene and embedded in Aquamount®. Tomato (Lycopersicon esculentum) lectin (Sigma, St Louis, MO, USA) was used for the staining of microglia, as follows. The frozen sections were melted for 20 min at room temperature, and then rinsed twice with 0.05 M Tris-buffered saline (TBS). Endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide in methanol for 15 min. After two rinses in TBS and a 15 min pretreatment with 0.3% Triton X-100 in TBS, the sections were incubated in biotin-labelled tomato lectin (3 µg/ml) in TBS with 0.3% of Triton X-100 for 2 h at room temperature, rinsed three times with TBS and then labelled with avidin peroxidase in TBS (1:30 000) for 1 h at room temperature. Diaminobenzidine (Sigma) in TBS was used as a chromogen for 5 min. Finally, the sections were rinsed with TBS and distilled water and embedded in Aquamount. An Olympus Vanox-T (Olympus, Inc., Tokyo, Japan) microscope, an Argus-10 (Hamamatsu Photonics, Hamamatsu City, Japan) image processor and a DT512N (Sony Precision Technology Inc., Japan) microcator were used for the morphometric measurements.
The volume of the vermian folia was measured using a point-counting method and the Cavalieri principle (Gundersen and Jensen, 1987). The corresponding area of each grid point a(p) was 0.055 mm2. Cryomicrotome settings were used to determine the section thickness (t = 10 µm). With a total magnification of x43, the volumes of folia II and X were measured. The numbers of grid points hitting the molecular
P(mo), granular
P(gr) and white matter
P(wm) layers were counted separately. The volumes were counted with the formula:
![]() |
The numbers of microglia were counted with the optical dissector method (Sterio, 1984; Gundersen, 1986
) at a magnification of x855 (objective x40). The average of the final section thickness was 5 µm, i.e. the thickness of the sections decreased by 50% during the histochemical processing. This means that when the dissector height used in the measurements was 3 µm, the actual dissector height (h) was 6 µm. The microglial nuclei, which were in focus on the reference section but not on the look-up section and were not touching the exclusion line (Gundersen, 1977
), were counted from the dissector frame of 10 000 µm2.
A in the formula below is the sum of the dissector areas. The sums of counted microglia in molecular
Q(mo), granular
Q(gr) and white matter
Q(wm) layers were measured separately starting at a random point and moving throughout the folia in a stepwise manner. The measurements were performed on systematically randomly selected sections and three sections in each animal were analysed. The numerical density of microglia was estimated with the formula:
![]() |
![]() |
Statistics
The results are expressed as means (± SD). Statistical analysis was performed by one-way analysis of variance (ANOVA) with the Statistical Package for the Social Sciences for Windows Software (7.5 release). Bonferroni-corrected t-tests were used for further comparing the group means and P < 0.0167 was considered a statistically significant difference (three parallel comparisons).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
At the end of the experiments the weights of the rats were 599 ± 35 g in the control group, 600 ± 48 g in the continuous group and 550 ± 32 g in the intermittent group. The difference between the groups was not significant [F(2,15) = 3.283, P = 0.066]. As previously reported, ANOVA for repeated measurements showed a significant difference in weight gain between the groups, the weight gain being smaller in the intermittent group (P < 0.001 for time x group interaction) (Riikonen et al., 1999).
The penetration of staining was complete and microglia were stained uniformly throughout the sections. As previously described, blood vessels and the ependyma were also tomato lectin positive (Acarin et al., 1994), but microglia were easily identified by their typical morphology (Figs 1 and 2
). Microglia were scattered throughout the cerebellum, but no microglial accumulations were seen. However, a number of microglia were found in the vicinity of blood vessels. No difference was observed between the groups in the general appearance or distribution of vermian microglia (Fig. 1
). Most of the microglia were ramified, and only a few amoeboid microglia were found in all groups. Ramified microglia have small cell bodies and long branched processes (Figs 1 and 2
). Amoeboid microglia resemble macrophages with large cell bodies and short processes.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Microglia are brain macrophages of monocytic origin (Ling et al., 1980). From a morphological point of view, microglia are divided into amoeboid and ramified. Ramified (resting) microglia represent the majority of microglia found in normal adult CNS. During brain degeneration, microglia become hypertrophic and phagocytic, and change their phenotype and morphology towards macrophages, to become amoeboid or reactive microglia (Streit et al., 1988
). The reactive microglia change back to the ramified form when the CNS damage is repaired (Giulian and Baker, 1986
; Suzumura et al., 1990
, 1991
). Only a few amoeboid microglia were found in the present study, and no statistical analysis on the number of reactive/amoeboid microglia could therefore be performed. It seems that the nature of ethanol-induced CNS lesion is slow, and only a few activated microglia are needed to phagocytose the damaged tissue at a time.
Microglia are activated and/or their number increases in several CNS diseases. In Alzheimers disease, microglia are associated with senile plaques (Perlmutter et al., 1990). In brain ischaemia, activated microglia phagocytose degenerated or dead neurons (Gehrmann et al., 1992
). In multiple sclerosis, microglia may present CNS antigens to circulating T-cells (Banati and Graeber, 1994
). The main targets of HIV-1 infection in the CNS are microglia and macrophages (Price et al., 1988
). When the cells are infected, they release cytotoxins to induce neuronal degeneration (Guilian et al., 1990). As shown in the present study, intermittent ethanol exposure also increases the number of cerebellar microglia; this finding was observed prior to the appearance of any signs of cerebellar atrophy. Microgliosis was seen in the anterior superior part of the vermis; in the same area where ethanol-induced cerebellar atrophy is most prominent in human alcoholics (Viktor et al., 1959
; Torvik and Torp, 1986
; Phillips et al., 1987
) and in experimental animals (Rintala et al., 1997
). It is therefore possible that microglia contribute to brain atrophy by phagocytosing degenerated CNS tissue. In line with this view, cerebral atrophy has been found to correlate strongly with cortical microgliosis in AIDS patients (Gelman, 1993
). At the time of undertaking the present study, our suggestion was that the changes are most likely to be found in the anterior superior (folium II) part of the vermis. In line with previous studies and according to our hypothesis, the ethanol-induced microglial changes were found in folium II, but not in the folium X.
Silver staining, immunohistochemistry and lectin histochemistry have been used to visualize microglia. Silver staining is rather an unspecific method labelling not only microglia, but also oligodendrocytes. Immunohistochemistry usually gives stronger labelling to amoeboid, than ramified, microglia (OX-42, OX-18 and RMG1) or does not stain ramified mircroglia at all (OX-41, OX-6, ED3 and anti-vimentin). Tomato lectin was used for microglial staining in the present study as it labels reliably both reactive and resting microglia. Lectins are proteins or glycoproteins of non-immune origin (Goldstein and Hayes, 1978; Alroy et al., 1988
). They have been isolated from many natural sources, including seeds, roots, bark, fungi, bacteria, seaweed, sponges, molluscs, fish eggs, body fluids of invertebrates and lower vertebrates and from mammalian cell membranes. Although the physiological functions of the lectins are unknown, they have a wide variety of applications in vitro. Their unique ability to recognize specific sugar residues of complex glycoproteins makes them highly valuable in cytological and histological studies for the identification of different cell types (Goldstein and Hayes, 1978
). The tomato lectin has been reported to specifically label both amoeboid and ramified microglia, as well as endothelial cells and ependyma, without any binding to neurons and other glial cells (Acarin et al., 1994
). Similar results were obtained in the present study. The specific morphology of microglia made their distinction from blood vessels and ependyma reliable.
In a previous study, performed on the same material as the present one, we found that a 51/2-month intermittent ethanol exposure decreased the number of peripheral sympathetic neurons by 28%, compared to the continuously ethanol-exposed group (Riikonen et al., 1999). Also in the cerebellum, binge ethanol exposure has been shown to induce more severe damage in neonatal rats, than continuous consumption (Bonthius and West, 1990
), and a loss of Purkinje cells has been reported after a single ethanol withdrawal in adult rats (Phillips and Cragg, 1984
). It has previously been suggested that ethanol-induced CNS degeneration is too slow to cause any noticeable microglial reaction (Streit, 1994
). This may be the case when ethanol exposure is continuous and chronic. But as we show in the present study, microgliosis is found in the cerebellum after intermittent ethanol exposure. Microgliosis probably reflects a more severe neuronal degeneration caused by binge ethanol consumption, compared to the degeneration caused by continuous ethanol exposure.
To date, only a few studies have been published on ethanol-induced alterations in microglia. Chronic ethanol treatment appears to accelerate the degeneration of microglial processes, and therefore accentuate the effects of normal ageing on microglia (Kalehua et al., 1992). However, no age-related changes were found in the number of microglia in the dentate gyrus, CA 1 or hilus of mouse hippocampus (Long et al., 1998
). Ethanol exposure has been found to increase superoxide anion production and to depress nitric oxide levels in cultured (resting) microglia (Colton et al., 1998
). This may result in an increased oxidative stress in the CNS and further degeneration of neurons, since the latter are particularly vulnerable to oxidative stress (Cohen and Warner, 1993
). Neuronal degeneration, in turn, may lead to an increased number of microglia and microglial activation. A putative pathogenic vicious circle with ethanol/withdrawal-induced neuronal degeneration and microglial alterations inducing each other is represented in Fig. 4
. Microglial functions have been found to deteriorate during chronic ethanol treatment in vitro, which may reflect an increased risk of CNS infections in vivo (Aroor and Baker, 1998
). The functions of microglia were beyond the scope of the present study, but CNS infections were not found post mortem in either the ethanol-exposed or the control rats.
|
In conclusion, intermittent ethanol exposure increases the number of microglia in the anterior superior part of rat vermis, which has been shown to be the most vulnerable part of the cerebellum to ethanol-induced degeneration. The microgliosis found in the present experiment occurred selectively in the molecular layer of folium II, in the same area where cerebellar atrophy was previously found after lifelong ethanol exposure (Rintala et al., 1997). The number of microglia increased before any volumetric changes of the cerebellar layers were found. Therefore, microglia may be used as early markers of ethanol-induced nervous system damage, before any macroscopic changes are discernible.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alroy, J., Ucci, A. A. and Periera, M. E. (1988) Lectin histochemistry: an update. In Advances in Immunohistochemistry, DeLellis, R. A. ed., pp. 93131. Raven Press, New York.
Aroor, A. R. and Baker, R. C. (1998) Ethanol inhibition of phagocytosis and superoxide anion production by microglia.Alcohol 15, 277280.[ISI][Medline]
Banati, R. B. and Graeber, M. B. (1994) Surveillance, intervention and cytotoxicity: is there a protective role of microglia?Developmental Neuroscience 16, 114127.[ISI][Medline]
Bautista, A. P. and Spitzer, J. J. (1992) Acute ethanol intoxication stimulates superoxide anion production by in situ perfused rat liver.Hepatology 15, 892898.[ISI][Medline]
Bautista, A. P. and Spitzer, J. J. (1999) Role of Kupffer cells in the ethanol-induced oxidative stress in the liver.Frontiers in Bioscience 4, 589595.
Bonthius, D. J. and West, J. R. (1990) Alcohol-induced neuronal loss in developing rats: increased brain damage with binge exposure.Alcoholism: Clinical and Experimental Research 14, 107118.[ISI][Medline]
Cohen, G. and Warner, P. (1993) Free radicals, oxidative stress and neurodegeneration. In Neurogenerative Disease, Calne, D. ed., pp. 139161. Saunders, Philadelphia.
Colton, C. A., Snell-Callanan, J. and Chernyshev, O. N. (1998) Ethanol induced changes in superoxide anion and nitric oxide in cultured microglia.Alcoholism: Clinical and Experimental Research 22, 710716.[ISI][Medline]
Gehrmann, J., Bonnekoh, P., Miyazawa, T., Oschlies, U., Dux, E., Hossmann, K. A. and Kreutzberg, G. W. (1992) The microglial reaction in the rat hippocampus following global ischemia: immuno-electron microscopy.Acta Neuropathologica 84, 588595.[ISI][Medline]
Gelman, B. B. (1993) Diffuse microgliosis associated with cerebral atrophy in the acquired immunodeficiency syndrome.Annals of Neurology 34, 6570.[ISI][Medline]
Giulian, D. and Baker, T. J. (1986) Characterization of ameboid microglia isolated from developing mammalian brain.Journal of Neuroscience 6, 21632178.[Abstract]
Giulian, D., Vaca, K. and Noonan, C. A. (1990) Secretion of neurotoxins by mononuclear phagocytes infected with HIV-1.Science 250, 15931596.[ISI][Medline]
Goldstein, I. J. and Hayes, C. E. (1978) The lectins: carbohydrate-binding proteins of plants and animals.Advances in Carbohydrate Chemistry and Biochemistry 35, 127340.[Medline]
Gundersen, H. J. G. (1977) Notes on the estimation of the numerical density of arbitrary profiles: the edge effect.Journal of Microscopy 111, 219223.[ISI]
Gundersen, H. J. G. (1986) Stereology of arbitrary particles: a review of unbiased number and size estimators and the presentation of some new ones, in memory of William R. Thompson.Journal of Microscopy 143, 345.[ISI][Medline]
Gundersen, H. J. G. and Jensen, E. B. (1987) The efficiency of systematic sampling in stereology and its prediction.Journal of Microscopy 147, 229263.[ISI][Medline]
Heinsen, H. and Heinsen, Y. L. (1984) Strain-specific differences in the vermian granular layer of albino rats.Acta Anatomica 119, 165173.[ISI][Medline]
Järveläinen, H. A., Oinonen, T. and Lindros, K. O. (1997) Alcohol-induced expression of the CD14 endotoxin receptor protein in rat Kupffer cells.Alcoholism: Clinical and Experimental Research 21, 15471551.[ISI][Medline]
Kalehua, A. N., Streit, W. J., Walker, D. W. and Hunter, B. E. (1992) Chronic ethanol treatment promotes aberrant microglial morphology in area CA1 of the rat hippocampus.Alcoholism: Clinical and Experimental Research 16, 401.
Karhunen, P. J., Erkinjuntti, T. and Laippala, P. (1994) Moderate alcohol consumption and loss of cerebellar Purkinje cells.British Medical Journal 308, 16631667.
Lees, G. J. (1993) The possible contribution of microglia and macrophages to delayed neuronal death after ischemia.Journal of the Neurological Sciences 114, 119122.[ISI][Medline]
Ling, E. A., Penney, D. and Leblond, C. P. (1980) Use of carbon labeling to demonstrate the role of blood monocytes as precursors of the ameboid cells present in the corpus callosum of postnatal rats.Journal of Comparative Neurology 193, 631657.[ISI][Medline]
Long, J. M., Kalehua, A. N., Muth, N. J., Calhoun, M. E., Jucker, M., Hengemihle, J. M., Ingram, D. K. and Mouton, P. R. (1998) Stereological analysis of astrocytes and microglia in aging mouse hippocampus.Neurobiology of Aging 19, 497503.[ISI][Medline]
Lukkari, T. A., Järveläinen, H. A., Oinonen, T., Kettunen, E. and Lindros, K. O. (1999) Short-term ethanol exposure increases the expression of Kupffer cell CD14 receptor and lipopolysaccharide binding protein in rat liver.Alcohol and Alcoholism 34, 311319.
Nakajima, K. and Kohsaka, S. (1993) Functional roles of microglia in the brain.Neuroscience Research 17, 187203.[ISI][Medline]
Nordmann, R., Ribiere, C. and Rouach, H. (1992) Implication of free radical mechanisms in ethanol-induced cellular injury.Free Radical Biology and Medicine 12, 219240.[ISI][Medline]
Perlmutter, L. S., Barron, E. and Chui, H. C. (1990) Morphologic association between microglia and senile plaque amyloid in Alzheimers disease.Neuroscience Letters 119, 3236.[ISI][Medline]
Phillips, S. C. and Cragg, B. G. (1984) Alcohol withdrawal causes a loss of cerebellar Purkinje cells in mice.Journal of Studies on Alcohol 45, 475480.[ISI][Medline]
Phillips, S. C., Harper, C. G. and Kril, J. (1987) A quantitative histological study of the cerebellar vermis in alcoholic patients.Brain 110, 301314.[Abstract]
Price, R. W., Brew, B., Sidtis, J., Rosenblum, M., Scheck, A. C. and Cleary, P. (1988) The brain in AIDS: central nervous system HIV-1 infection and AIDS dementia complex.Science 239, 586592.[ISI][Medline]
Riikonen, J., Jaatinen, P., Karjala, K., Wu, X., Pörsti, I. and Hervonen, A. (1999) Effects of intermittent versus continuous ethanol exposure on rat sympathetic neurons.Alcoholism: Clinical and Experimental Research 23, 12451250.[ISI][Medline]
Rintala, J., Jaatinen, P., Lu, W., Sarviharju, M., Eriksson, C. J. P., Laippala, P., Kiianmaa, K. and Hervonen, A. (1997) Effects of lifelong ethanol consumption on cerebellar layer volumes in AA and ANA rats.Alcoholism: Clinical and Experimental Research 21, 311317.[ISI][Medline]
Shiratori, Y., Teraoka, H., Matano, S., Matsumoto, K., Kamii, K. and Tanaka, M. (1989) Kupffer cell function in chronic ethanol-fed rats.Liver 9, 351359.[ISI][Medline]
Sterio, D. C. (1984) The unbiased estimation of number and sizes of arbitrary particles using the disector.Journal of Microscopy 134, 127136.[ISI][Medline]
Streit, W. J. (1994) Microglia in the pathological brain. In Alcohol and Glial Cells, Lancaster, F. E. ed., pp. 5567. NIH, Bethesda, Maryland.
Streit, W. J., Graeber, M. B. and Kreutzberg, G. W. (1988) Functional plasticity of microglia: a review.Glia 1, 301307.[ISI][Medline]
Suzumura, A., Sawada, M., Yamamoto, H. and Marunouchi, T. (1990) Effects of colony stimulating factors on isolated microglia in vitro.Journal of Neuroimmunology 30, 111120.[ISI][Medline]
Suzumura, A., Marunouchi, T. and Yamamoto, H. (1991) Morphological transformation of microglia in vitro.Brain Research 545, 301306.[ISI][Medline]
Tavares, M. A., Paula-Barbosa, M. M. and Cadete-Leite, A. (1987) Chronic alcohol consumption reduces the cortical layer volumes and the number of neurons of the rat cerebellar cortex.Alcoholism: Clinical and Experimental Research 11, 315319.[ISI][Medline]
Thurman, R. G. (1998) Alcoholic liver injury involves activation of Kupffer cells by endotoxin.American Journal of Physiology 275, 605611.
Torvik, A. and Torp, S. (1986) The prevalence of alcoholic cerebellar atrophy. A morphometric and histological study of an autopsy material.Journal of the Neurological Sciences 75, 4351.[ISI][Medline]
Viktor, M., Adams, R. and Mancall, E. L. (1959) A restricted form of cerebellar cortical degeneration occurring in alcoholic patients.Archives of Neurology 1, 579688.[ISI]