Limitations of monoclonal antibodies for monitoring of fungal aerosols using Penicillium brevicompactum as a model fungus.

Molds are ubiquitous in every environment and many species have been recently associated with an increase in opportunistic infections in immunocompromised patients or the exacerbation of asthmatic episodes in allergic patients. The degree of environmental contamination with fungi thus needs to be monitored and in this study we report the development of a monoclonal antibody (mAb)-mediated enzyme-linked immunosorbent assay (ELISA) for the detection of spores of Penicillium brevicompactum in experimental model aerosols. In addition, we have investigated the influence of different parameters of air sampling and sample recovery on ELISA performance. MAbs were produced with standard hybridoma techniques and cross-reactivities were determined against spores of 53 fungal species by indirect ELISA. Standardized experimental fungal aerosols were collected with the Button Personal Inhalable Aerosol Sampler onto polycarbonate or polytetrafluoroethylene filters (PTFE) and the effects of different extraction buffers and filter agitation methods during sample processing on spore recovery and ELISA detection were investigated. Five mAbs were produced and all of them cross-reacted with several of 31 related Aspergillus, Penicillium and Eurotium species. However, cross-reactivities with 21 non-related fungi were rare. Spores were recovered in much higher numbers from polycarbonate filters (PFs) than from polytetrafluoroethylene filters. Optical densities (ODs) in ELISA were higher for spores collected into carbonate coating buffer (CCB) than phosphate-buffered saline (PBS). Filter bath sonication following filter vortexing had no positive effects on ELISA sensitivity. The cross-reactivity patterns of mAbs suggest that Aspergillus and Penicillium species share multiple antigens. Quantitative ELISA results for fungal aerosols were found to be influenced by differential sample processing and thus method standardization will be essential to maintain the comparability of immunometric monitoring results.

Long-term behavioural effects of a repeated exposure to chlorphenvinphos in rats.

The aim of the present study was to investigate persistent neurobehavioural effects of repeated low-level exposure to chlorphenvinphos ((2-chloro-1-(2,4-dichlorophenyl) vinyl diethyl phosphate-CVP) in rats. The rats received 10 i.p. injections of CVP at daily doses of 0.5 mg/kg or 1.0 mg/kg (one injection/day, five days/week) which corresponded to 1/20 and 1/10 of LD50, respectively, for this species. In a part of the rats, cholinesterase (ChE) activity in blood (plasma and erythrocytes), and in the selected brain regions was determined at arbitrarily chosen time after the last exposure. The determinations showed that the level of ChE inhibition was dose-related, but the compartments studied differed in the magnitude of this effect. The differences in the level of ChE inhibition between the compartments were particularly evident in rats which had received CVP at the 1.0 mg/kg dose; in these animals 3 h after exposure the ChE activity in erythrocytes, plasma and the brain corresponded to 78%, 48% and 67-70%, respectively, of the control value. Enzyme activity returned to the control level after 14 days in plasma and after 35 days in erythrocytes. In rats receiving CVP at daily doses of 0.5 mg/kg, ChE activity in plasma was decreased by 40.8% and that in erythrocytes by 21.4% 3 h after the last exposure. The activity of ChE in plasma returned to the control level within four days and that in erythrocytes within 14 days. In these rats, in all the brain regions studied except brainstem, ChE activity was not reduced significantly. In rats selected for behavioural tests, the following behavioural aspects were investigated: response to novelty in an open field, acquisition and extinction of a one-way active avoidance response, and the magnitude and persistence of the footshock-induced analgesia (hot-plate test). Testing in the open field was performed before the exposure and then 1, 3 and 6 weeks after the last exposure. The remaining tests were performed after the exposure. The interval between testing and the last CVP injection was sufficient for recovery of ChE activity. It has been found that in rats of both exposure groups the response to novelty in the open field, i.e. the increase in locomotor and exploratory activity in the presence of a new object, was reduced, albeit nonsignificantly, compared to the unexposed animals. In rats which received CVP at the dose of 1.0 mg/kg, acquisition of the one-way active avoidance response was facilitated. No differences between groups were found during extinction of this response. In the hot-plate test, in rats exposed repeatedly to 1.0 mg/kg CVP, the footshock-induced increase in the latency of the paw-lick response to heat (54.5 degrees C) was stronger and more persistent than in the unexposed animals. The above results show that some neurobehavioural effects of exposure to organophosphorous (OP) compounds may be detected after a time sufficient for recovery of ChE activity.

Application of the classic Limulus test and the quantitative kinetic chromogenic LAL method for evaluation of endotoxin concentration in indoor air.

The classic (gel-clot procedure) Limulus test (CLT) and the quantitative kinetic chromogenic LAL method (KQCL) used for the evaluation of bacterial endotoxin concentration in the indoor air of dwellings were compared. The scientific procedure included analyses of 40 air samples supplemented by the analysis of 20 sample duplicates (selected at random) which were taken during the fall season from 10 flats located in 3 towns of the Upper Silesian region (southern Poland). The particulate aerosol probes were sampled by Harvard impactor and Casella sampler. The same samples were analyzed in the Netherlands using the quantitative kinetic chromogenic LAL method, and in Poland using the classic Limulus test. Comparison of both methods revealed that the quantitative kinetic chromogenic LAL method was more precise, with better reproducibility (the coefficient of variation between analyses of the main probe and its duplicate was over two times smaller in the KQCL method than in the CLT method), fully automated in the phase of analysis and data reading, and faster and more effective than the classic Limulus test. Nevertheless, on the basis of the obtained results, the usefulness of the classic Limulus method for assessment of the degree of pollution of indoor air with bacterial endotoxin seems to be confirmed as in the majority of examined samples (21 out 40) the results obtained by both methods were of the same order of magnitude, and in the remaining 19 samples did exceed one order of magnitude. Thus, the data received by means of the classic Limulus test may be regarded as acceptable.

[The prevalence of bacterial and fungal aerosol in homes, offices and ambient air of Upper Silesia. Preliminary results]. / Wystepowanie aerozolu bakteryjnego i grzybowego w mieszkaniach, biurach i srodowisku zewnetrznym Górnego Slaska. Wyniki Wstepne.

Quantitative criteria of microbiological air quality in homes and offices are needed for practical reasons. The purpose of this study was to obtain the preliminary characteristic of bacterial and fungal aerosols in healthy buildings. It was analysed the concentrations levels and size distributions of the investigated bioaerosols. The obtained data can be treated as a first step in the direction of determining so called normal level for different bioaerosols in homes and offices in Poland. The concentrations of airborne bacteria and fungi were measured using 6-stage Andersen impactor. The Trypcase Soy Agar were applied for bacteria and 2% Malt Extract Agar for fungi. The bacteria samples were incubated for 2 days at 37 degrees C and the fungi samples respectively for 4 days at 25 degrees C. The indoor levels of bacterial aerosol (homes: 212-888 cfu/m3, offices: 136-542 cfu/m3) were higher than the outdoor levels (respectively: 42-386 cfu/m3 and 13-115 cfu/m3). The fungal aerosol concentrations were lower indoors (homes: 81-383 cfu/m3, offices: 18-133 cfu/m3) than outdoors (94-625 cfu/m3). There were distinctly higher concentrations of the investigated bioaerosols in homes than in offices. The aerodynamic diameter of most bacterial particles were higher than 7 microns, both in indoor air (homes: 57-260 cfu/m3, offices 19-118 cfu/m3) and outdoor air (respectively: 9-145 cfu/m3 and 0-71 cfu/m3). The maximum for fungal spore levels were observed in the size range 3.3-4.7 and 2.1-3.3 microns in the instance of homes (respectively: 12-155 cfu/m3 and 19-154 cfu/m3) and in the size range 2.1-3.3 microns in the instance of offices (6-55 cfu/m3). Largest numbers of this bioaerosol in outdoor air were isolated in the size range 2.1-3.3 microns (25-208 cfu/m3). Although there are some proposals for an upper limit of the normal indoor concentration of airborne bacteria and fungi, but due to different climate and housing conditions we can expect other normal range of fungal and bacterial aerosol in Polish homes.

[Dust particles and metals in outdoor and indoor air of Upper Silesia]. / Czastki pylu i metale w powietrzu zewnetrznym i w pomieszczeniach na Górnym Slasku.

This work contains the results of the aerosol mass size distribution and preliminary studies on concentrations and size distribution of heavy metals (Pb, Zn, Cu, Mn, Fe and Cd) in indoor and outdoor environment in Upper Silesia (the highly industrialized region in the southern part of Poland). In studies, the measurements of aerosol concentration, mass size distribution, and evaluation of heavy metals concentration were made from December 1992 to April 1994 in some apartments in five towns in Upper Silesia and in one village in the Beskidy Mountains in both indoor and outdoor environments. The particles were fractionated in Andersen cascade impactor. The sampling time was 6-7 days and 4-5 days for indoor and outdoor respectively. Aerosol particulates were collected on A-type glass fiber collection substrate used later for determination of heavy concentrations by atomic absorption spectrophotometry (AAS 3, Carl Zeiss Jena). The dust was mineralized by the means of the mixture of hydrofluoric and nitric acids. The results of mass size distribution as well as the measurements of TSP for indoor and outdoor aerosol show that the main source of particulate matter indoors, in this region, are heavy polluted outdoor air and cigarette smoking. It can be said that, except homes in Knurów and Sosnowiec with hard smokers, the indoor levels of particulate pollution were significant lower than the outdoors levels. Whenever in the indoor environment appear additional source of particulate emission situation can changed. When we compare mass size distribution for outdoor aerosol and indoor aerosol contaminated by tobacco smoke, we can observed considerable increase of indoor aerosol level in the 0.33-0.54 microns size range. Besides, indoor aerosol status may be changed by coal stove emission (displacement of maximum peak to direction of coarse particles). The observed differences in concentration of particulate matter may also indicate the important differences in chemical and physical nature of particles caused by the air filtration and absorption during migration of ambient air into the indoor environment. On the base of comparison of the heavy metals concentrations of fine and coarse fraction and their indoor/outdoor ratio in five selected towns in Upper Silesia it can be said that the level of heavy metals in indoor aerosol is lower than in outdoor (except Pb and Cd) what suggest that migration of ambient air into the homes is a major process which give indoor air contamination of heavy metals.(ABSTRACT TRUNCATED AT 400 WORDS)

Subacute oral toxicity of 2-butyne-1,4-diol in rats.

2-Butyne-1,4-diol was given to male and female Wistar Imp:DAK rats by oral gavage for 28 consecutive days in daily doses of 1, 10 or 50 mg kg-1 day-1. After 28 days all animals were necropsied. Blood samples were obtained and selected organs were weighed and prepared for histological examination. Treatment-related effects in the high-dose group consisted of: fatal cases in both sexes; depressed body weight gain in males; increase of absolute and/or relative weights of liver and kidneys in both sexes; decreased red blood cell count, haematocrit value and haemoglobin concentration in female rats and elevated reticulocyte count and leukocyte count in both sexes; increased total serum protein content in females, elevated glucose concentration in males and higher activity of sorbitol dehydrogenase in both sexes; and histopathological evidence of hepatotoxicity and nephrotoxicity in decedents, and hepatic and splenic changes in survivors. Minor hepatic, splenic and erythrocytic changes were also found in some females given the middle dose. The dose of 1 mg kg-1 day-1 was considered to be the no-observed-effect level (NOEL), and 10 mg kg-1 day-1 the lowest-observed-effect level (LOEL).

Toxic effects of combined exposure to toluene and m-xylene in animals. III. Subchronic inhalation study.

Effects of combined exposure to toluene and m-xylene in the conditions of subchronic inhalation experiments in rats were examined. Rats were exposed to vapours of individual solvents and their 1:1 mixture at concentrations of 1000 ppm or 100 ppm, 6 h/day, 5 days/week, for 3 or 6 months, respectively. In rats exposed for 3 or 6 months to toluene, m-xylene and their mixtures (1:1) at concentrations of 1000 ppm and 100 ppm, respectively, the observed disturbances in rotarod performance test and decrease in spontaneous motor activity were statistically significant in comparison to control. In animals exposed to mixtures (1:1) of toluene and m-xylene, changes were not significantly different but more pronounced when compared to single solvent groups. The decrease of red blood cells count and increase of rod neutrophil cell counts were observed only in rats exposed for 3 months to mixture of solvents. Results obtained in condition of acute and subchronic inhalation exposure and toxicokinetics data interpreted jointly indicate the more than additive toxic effects of combined exposure to toluene and m-xylene.

Tolerance to chlorphenvinphos in rats assessed on the basis of changes in locomotor behavior in rotating wheels.

Spontaneous locomotor activity in rotating wheels was investigated in rats exposed repeatedly (i.p. daily injections, five days a week for two weeks) to an agricultural organophosphorus pesticide, chlorphenvinphos (CVP) at doses of 1.0 and 3.0 mg/kg. After a seven day interval each rat was injected with a single 3.0 mg/kg test dose of CVP in order to assess the stability of tolerance. Concomitant changes in blood and the brain ChE activity were also investigated. It was found that exposure to CVP at a low dose (1.0 mg/kg), resulting in less than 50% reduction of ChE activity in blood and in the brain, did not produce changes in spontaneous locomotion in rotating wheels in the rat. Higher doses (3.0 mg/kg) inhibited blood and the brain ChE by more than 50% and reduced locomotion. Under conditions of repeated exposure to CVP at the symptomatic (3.0 mg/kg) dose ChE activity remained low throughout the exposure period, however, locomotor activity returned to a normal level, i.e. tolerance developed, within less than five days. Seven days after termination of the repeated exposure, the behavioral subsensitivity to CVP still remained. The biochemical data suggest that it may be related, at least partially, to a diminished vulnerability of ChE in some parts of the brain to CVP induced inhibition.

Changes in brain bioelectrical activity (EEG) after repetitive exposure to an organo-phosphate anticholinesterase. II. Rat.

Effects of repetitive exposure (ten times in a period of two weeks) to chlorphenvinphos (CVP), at daily doses of 0.5 and 1.0 mg/kg, i.p., were studied in adult male Wistar rats of imp-DaK stock. It was found that 3 hrs after the last exposure, the cholinesterase (ChE) activity in the blood and brain was close to 50% of the control value in the 0.5 mg/kg group and less than 50% in the 1.0 mg/kg group. In both groups, normalization of ChE activity in plasma took less time than in erythrocytes, and the normalization of ChE activity in erythrocytes proceeded faster than in the majority of the brain areas studied. Electrophysiological investigations revealed a retardation of age-related epileptic-like cortical activity. This effect, however, was present only in the 1.0 mg/kg group, and only in the period of decreased ChE activity in the brain. Spectral analysis revealed an increase in 1-4 Hz activity in the cortical EEG of the 0.5 mg/kg group and heightened theta activity (4-7 and 7-9 Hz bands) in the hippocampal EEG of the 1.0 mg/kg group. The later effects were detected after a time sufficient for full normalization of ChE activity and manifested themselves most clearly in the presence of an acoustic stimulus associated with pain. The above results are in agreement with earlier observations on rabbits exposed repetitively to CVP. Data from both species suggest that, in the case of repetitive exposure to CVP, neither plasma nor erythrocyte ChE activity is a reliable indicator of toxicity, and that such exposure to this OP may lead to changes in EEG outlasting the period of lowered ChE activity in the blood and brain.

Brain electrical activity (EEG) after repetitive exposure to chlorphenvinphos an organophosphate anticholinesterase: I. Rabbit.

Cholinesterase (ChE) activity in blood and brain as well as the hippocampal and cortical EEG were investigated in rabbits exposed once a day for a period of two weeks to an organophosphate insecticide, chlorphenvinphos (CVP). The daily dose of CVP was 14.0 mg/kg i.p. ChE activity in plasma and erythrocytes decreased by 60 and 48%, respectively, by the end of exposure and returned to the preexposure level within 35 days. In the exposed animals, killed and dissected a day after, the ChE activity in blood had been found normal, the level of ChE activity in some parts of the brain was still significantly depressed. The spontaneous hippocampal EEG showed no changes as soon as 24 hours after the exposure. However, after a period sufficiently long for the normalization of ChE activity in blood, the hippocampal arousal response (theta rhythm) to click and to a tone associated with pain was found heightened in the exposed rabbits. Moreover, spectral analysis of 5 minute EEG samples revealed a decrease in the content of 7-13 Hz activity in the cortex of the exposed animals as compared to the control ones. The obtained data suggest that exposure to CVP may lead to functional changes in the brain outlasting the period of ChE depression.

Subacute oral toxicity of 1,4-butanediol in rats.

1,4-Butanediol (BAD) was administered to male and female Wistar Imp:DAK rats by oral gavage for 28 consecutive days. Treated rats received BAD at daily doses of 5, 50 or 500 mg/kg/day. After 28 days all animals were necropsied. Blood samples were obtained and selected organs were weighed and prepared for histological examination. Subacute oral administration of BAD resulted in an overall low degree of systemic toxicity. There were no changes in body weight, food consumption, and absolute and relative organ weights. Slightly higher activities of sorbitol dehydrogenase and alanine aminotransferase were observed in male rats given BAD at the highest dose of 500 mg/kg/day. Some disturbances in hematological parameters, characterized by macrocytosis and thrombocytopenia were observed in treated rats. Mild to moderate inflammation of the liver, characterized by proliferation of bile ducts and periportal infiltrations with fibroblasts and mononuclear cells, were found in treated animals. A statistically significant difference for histopathological changes was found in animals treated with BAD at the dose of 500 mg/kg/day only in the case where both sexes were jointly taken for comparison.

Changes in physiological and electrophysiological parameters in rabbits after single exposure to chlorphenvinphos.

In order to evaluate some of the central effects of an organophosphorus pesticide--chlorphenvinphos (CVP), the time course of changes in the activity of blood cholinesterase (ChE), body temperature and hippocampal EEG were compared in rabbits after acute i.p. exposure. The pesticide was administered twice at an interval of 80-90 days. The Deichmann-LeBlanc scheme of dosing (one animal/one dose) was adopted starting with 22 mg/kg. CVP resulted in a dose-dependent decrease in plasma and erythrocyte ChE activity, a decrease in body temperature ranging from 0.7 degrees to 3.5 degrees C, and in an increase in content of the immobility-related, rhythmic slow activity (I-RSA) in the hippocampal EEG. Changes in body temperature appeared at lower doses than those in the hippocampal EEG. Contrary to the changes in ChE activity, which lasted 4-30 days, those in body temperature and in hippocampal EEG disappeared within 24 hours after the injection. CVP administered at the same dose 80-90 days after the first injection, resulted again in an inhibition of ChE activity, but the effect on the hippocampal EEG was less clear, and that on body temperature was variable; no effect, an increase or a decrease appeared. The data suggest that: i) body temperature is a more sensitive index of a central action of CVP administered for the first time than the hippocampal EEG; ii) the brain cholinergic mechanisms are relatively resistant to the acute action of the OP and undergo fast adaptive changes; iii) even single exposure to CVP may produce some long-lasting functional changes in the brain of some subjects, which has been proven by the changed response to the second exposure.

Neurotoxicity of chlorphenvinphos an organophosphorus pesticide: effects on blood and brain cholinesterase activity, open field behavior and response-to-change in a "T" maze in rats.

Cholinesterase (ChE) activity in blood (plasma and erythrocytes) and in different parts of the brain, open field behavior and response-to-change in a "T" maze were investigated in separate groups of rats after a single intraperitoneal exposure to an organophosphorus pesticide - chlorphenvinphos. The doses used were 3.0 mg/kg or 1.0 mg/kg which amounted to approximately, 1/3 and 1/10 of LD50 for this species. The exposure resulted in a dose dependent inhibition of ChE in blood, as well as in the brain. No marked differences in the level of ChE inhibition in plasma, erythrocytes, and in selected parts of the brain (cerebellum, brain stem, diencephalon, hippocampus and the anterior part of the hemisphere) were noted after a given dose. In general, 3 hours after the exposure, ChE was inhibited by about 80% in the case of the 3.0 mg/kg dose and by no more than 50% in the case of the 1.0 mg/kg dose. In blood as well as in the brain, the normalization of ChE activity proceeded at a similar rate, being accomplished within 14 days and within 94 hours after the 3.0 mg/kg and 1.0 mg/kg dose, respectively. No changes suggesting an impairment of short-term memory were observed in the "T" maze until the end of testing (i.e. up to the fourteenth day after the exposure). In the open field, a short-term decrease (up to the third day) in locomotor and exploratory activity was observed only in rats exposed to 3.0 mg/kg of the pesticide. At the end of testing (10-14th day after the exposure) the introduction of a new object into the open field resulted in an increase of locomotor and exploratory activity in control animals but not in the rats exposed to chlorphenvinphos in the 3.0 mg/kg as well as in the 1.0 mg/kg. This suggests that exposure to chlorphenvinphos may result in some behavioral disturbances lasting longer than the ChE recovery.

[Experimental studies of acute and chronic toxic effects of vinyl acetate]. / Badania doswiadczalne nad ostrym i przewleklym dzialaniem toksycznym octanu winylu.

In view of divergent opinions concerning MAC value for vinyl acetate, a study on acute and chronic inhalatory toxic effect of this compound on animals was carried out. The scope of the study included determination of CL50 value after Litchfield and Wilcoxon and 10-month exposure of animals to vinyl acetate at concentrations 10, 100 and 500 mg/m3, 5 days weekly, 5 hours daily. During 10-month experiment the animals were observed and body weight controlled. In addition, periodically some hematological examinations and biochemical of blood serum as well as histopathological examinations of inner organs were carried on. Post-mortally the weight of inner organs was determined. CL50 value determined on rats has amounted to 4100 ppm. In the study on chronic effect, the prevalence and the degree of intensity of the changes of the used by us indicators was the least in the group of animals exposed to vinyl acetate at concentration of 10 mg/m3. These changes were transient not involved reticulopenia and animals' body weight decrease. Histopathological examinations revealed some inflammatory changes in respiratory system both in control and exposed animals. Only planoepithelial metaplasia of bronchi was found exclusively in animals exposed to vinyl acetate at all concentrations used. The changes within liver were found only in animals exposed to vinyl acetate at concentration of 100 and 500 mg/m3. These changes involved fatty degeneration of hepatic parenchyma, proliferation and extension of smooth endoplasmatic reticulum and the changes within the biliary canaliculi. Taking the above results into account it seems that the lowest of the recommended in the world MAC value--10 mg/m3 may be accepted as the upper limit of the maximum admissible concentration for vinyl acetate.

The fluorescent immunosorbent test for IgG gliadin antibodies and the leucocyte migration inhibition test in coeliac disease; comparison of diagnostic value.

The diagnostic value of the fluorescent immunosorbent test for IgG gliadin antibodies (FIST) has been investigated in comparison with the LIF test--the competence of the gluten subfractions B2 and B3 in releasing lymphokines from peripheral lymphocytes in vitro--in 96 patients with coeliac disease (CD) under various dietary conditions. In untreated children with CD during their first 2 years of life, the FIST showed 100% sensitivity with 95% specificity, whilst the LIF test showed only 70% sensitivity and 73% specificity. Therefore it can be concluded that the FIST as a screening test is superior to the LIF. In older children with a proved recurrence of the mucosal abnormality after reintroduction of a normal diet, only 44% showed increased IgG gliadin antibody titres whereas 70% proved to be positive in the LIF test. Under a controlled gluten challenge all six patients reacted with a distinct increase in gliadin antibody titres whereas the LIF test changed from positive to negative and vice versa without following any clear principle. These results emphasize the inadequacy of the LIF test as a diagnostic method, both in untreated CD and under controlled gluten challenge.

Effect of different conditions of acute exposure to carbon monoxide on the cerebral high-energy phosphates and ultrastructure of brain mitochondria in rats.

Rats were exposed to carbon monoxide (CO) in different conditions: 4 min at 1.3% CO, 40 min at 0.5% CO or 12 h at 0.13--0.15% CO. After 4 min exposure to 1.3% CO the brain content of ATP and PC was substantially reduced; after 40 min exposure to 0.5% CO the cerebral ATP level was slightly increased, whereas the content of both ATP and PC in the brain of rats exposed to CO for 12 h was significantly higher than in the controls. The decrease in the brain level of ATP and PC after 4 min exposure to 1.3% CO was accompanied by ultrastructural changes of mitochondria. No evident differences in the level of cerebral high-energy phosphates were found between rats intoxicated with CO and rats subjected to experimental hypoxemia.

The content of high-energy phosphates and ultrastructure of mitochondria in the brain of rats exposed to carbon disulphide.

Rats were exposed to carbon disulphide (CS2) continuously for 12 h or for 10 months, at concentrations of 2.4 mg/l air and 0.8 mg/l air, respectively. No changes in the brain content of ATP, ADP and AMP were found in rats after chronic exposure, whereas the content of ATP in the brain of rats acutely poisoned with CS2 was significantly higher (21%) accompanied by decrease in the content of ADP (10%) and AMP (45%). The biochemical changes were accompanied by ultrastructural changes of mitochondria, particularly those in the cerebellar perikaryons.