In the present study, a rat model of subacute methyl mercury (MeHg) intoxication was used to investigate possible MeHg-induced detrimental effects on cerebellar cortices. Thirty six adult male Sprague Dawley rats (aged 6 weeks) were randomly assigned into three equal groups viz. control group (group I), low methyl mercury exposure group (group II - 2.5 ppm) and high methyl mercury exposure group (group III - 5 ppm). Exposed rats were sacrificed at 14 and 35 days and cerebellar tissue samples were collected and examined the effect of the two levels of MeHg exposure for varying periods. Histological study of these samples revealed a reduced average thickness of molecular cell layer, granular cell layer, average number and size of Purkinje cells in both the treated groups and periods. Purkinje cell shape was altered in both exposure periods in 5 ppm group and loss of nucleoplasm and karyolysis in 2.5 ppm at 35 days post-exposure. Further, the granule cells were disoriented and a few altered their shape in both the treatment groups at 35 days post-exposure.
Mercury (Hg) is considered as a devastating environmental pollutant, mainly after the environmental disaster at Minamata (Japan) and several other poisoning accidents due to the use of Hg pesticides in agriculture (Morcillo et al., 2017). The safety limits for mercury in drinking water is 0.001 mg per liter in india with no relaxation. However, mercury levels in Indian rivers and Arabian Sea were found to be at alarming levels and Methyl mercury is one of the potent neurotoxin that stands as the sixth most serious pollution threat to the planet (Toxics Link, 2003). Once in the aquatic food chain, methyl mercury bio-accumulates and magnifies, reaching high levels in predatory fish, thus representing a toxicological concern for humans subsiding on fish for their dietary intake (Hintelmann, 2010; WHO, 2007).
Methyl mercury is the most toxic form of mercury in the environment (Clarkson and Magos, 2006) and despite its vast distribution among several tissues after absorption, the central nervous system (CNS) represents the main target of its toxicity, especially when exposures occur during early stages of neurodevelopment (Farina et al., 2011). The primary target organ for oral exposure to Methyl mercury was the brain, the effects on this organ accounting for the developmental toxicity of the chemical (Takahashi et al., 2017). It can easily cross the blood-brain barrier and placenta and is accumulated in the brain of fetuses at a greater degree compared to dams (ATSDR, 2012; Pereira et al., 2016). Studies have ascertained that Methyl mercury, a relevant persistent environmental contaminant is widely recognized as a potent neurotoxicant in humans affecting both developing and mature central nervous system (Sunol et al., 2012). Histological examination embraces the study of the structures of both tissue and cells, and the relationship between these structures and physiological functions; besides, it has been increasingly recognized as a valuable tool for field assessment of the impact of environmental pollutants on the livelihood (Musumeci, 2014). Hence, the presented histological work was conducted with an objective to understand the possible detrimental effects of methyl mercury at various levels on the cerebellar tissue of Sprague Dawley rats, in order that these findings can be applied in assessment of methyl mercury impacts on either human or wild life.
Materials and Methods
The experimental animal protocol followed the ethical principles as approved by the Institutional Animal Ethics Committee approved by CPCSEA (through reference 4/IAEC/NTRCVSc/GVM-2013-14 dated 10.12.13). The work was carried out at the Experimental laboratory animal house and Department of Veterinary Anatomy, N.T.R. College of Veterinary Science, Gannavaram, Andhra Pradesh, India.
Establishment of Experimental Rats
Thirty six adult male Sprague Dawley rats of 5 weeks age were procured from National Centre for Laboratory Animal Sciences, National Institute of Nutrition, Hyderabad, India. After quarantine for a week, subjects were randomly assigned into three equal groups viz. control group (group I), low Methyl mercury group (group II) and high Methyl mercury group (group III) (Table 1). The groups II and III were exposed to 2.5 ppm and 5 ppm of MeHg in the form of Methyl Mercuric Chloride (Sigma Aldrich 33368) in drinking water adlibitum daily. All the three groups were fed commercial pelleted rat diet (chow) adlibitum daily and were maintained under standard conditions of light (12/12-h light/dark cycle) and room temperature (22 ± 2ºC) as per CPCSEA norms. Six animals in each group were sacrificed humanely under spinal dislocation after 14 and 35 days of exposure to study the early and prolonged exposure effects (Table 1).
Table 1: Experimental Design
|Treatment Groups||Number of Animals Sacrificed|
|14 days post- exposure||35 days post-exposure|
|Control (Group I)||6||6|
|2.5ppm (Group II)||6||6|
|5.0ppm (Group III)||6||6|
After each sacrifice, fresh brains were collected from the cranial cavity from which cerebellum was separated, and half of the cerebellum from each animal was transferred to 10% neutral buffered formalin for histological studies and the rest halves were used for frozen sections.
The tissues for histological studies were processed and sections of 4-5µ thickness were stained with various staining methods (Luna, 1968), (Brancroft and Gamble, 2003) for astrocytes and glial cells (Sheehan et al., 1980) and (Crookham et al., 1991) for myelin to elucidate the histological architectural changes in cerebellar cortices of MeHg exposed rats.
Results and Discussion
Photomicrograph (PTAH 100X, Luxol Fast Blue 400X, and Trichrome 400X) showing the architecture of cerebellar cortex in control, 2.5 ppm and 5 ppm at 14- and 35- days post-exposure are presented in Fig. 1, 2, 3, 4, 5, 6, 7, 8, and 9 respectively.
Influence of Varying Levels of MeHg on Molecular layer of Cerebellum
The influence of varying levels of MeHg on the thickness of the molecular layer at 14- and 35- days post-exposure is presented in Table 2. The average thickness of the molecular cell layer was reduced in both the treated groups when compared to control group in the two treatment periods. These findings were similar to the reports of Hunter and Russell (1954) in humans exposed to industrial MeHg compounds.
Table 2: Average thickness of molecular layer in foliae of cerebellum on MeHg exposure
|Group||14 d post-exposure||35 d post-exposure||14 d post-exposure||35 d post-exposure||14 d post-exposure||35 d post-exposure|
|Control||64.8 µm||71.2 µm||66.8 µm||61 µm||65.4 µm||48 µm|
|2.5ppm||62.8 µm||61.8 µm||58.2 µm||58.2 µm||52.6 µm||41.2 µm|
|5ppm||43.6 µm||48.2 µm||55 µm||52.2 µm||46.8 µm||42 µm|
Many of the Purkinje cells lost their dendritic arborization in the molecular layer but were connected to granule cell layer in 2.5 ppm group at 14 days of exposure (Fig. 1).
|Fig. 1: Photomicrograph showing cerebellar circuitry in 2.5 ppm at 14 days post-exposure ( Luxol Fast Blue 400X)||
Fig. 2: Photomicrograph showing cerebellar circuitry in control at 35 days post-exposure (Luxol Fast Blue 400X)
Dendritic trees of purkinje cells were disorganized and disoriented. Main dendrite from the soma of some purkinje cells was detached and thrown in the molecular layer of 2.5 ppm treated group and was further increased in 5 ppm group at 35 days post- exposure (Fig. 2 & 3). The observed phenomenon was in consistent with the findings of Roegge et al. (2004) and Heath et al. (2010). Spongiosis in molecular layer and vacuolation between molecular layer and purkinje cell layer was observed in 5 ppm group at 14 days of exposure (Fig. 4). Spongiosis was amplified due to the vacuoles around the basket cells of 5 ppm group than in 2.5ppm group at 35 days of exposure. Similarly, Husain (2014) observed spongiosis in molecular layer of albino rats treated with mercuric chloride @ 0.33 mg/kg b.wt. No reports were evidenced to substantiate the findings pertaining to Basket cells, stellate cells and increased haemorrhages in the molecular layer of MeHg treated rats.
Influence of Varying Levels of MeHg on Purkinje Layer of Cerebellum
Average number of purkinje cells per foliae of cerebellum in different groups of rats is presented in Table 3.
Table 3: Average number of purkinje cells per foliae of cerebellum in different groups of rats
|14 days post- exposure||58||54||51|
|35 days post- exposure||71||60||54|
The average number and size of Purkinje cells was reduced in both the treatment groups at 14 and 35 days post-exposure (Fig. 4, 5 & 6).
Fig. 3: Photomicrograph showing cerebellar circuitry in 5 ppm at 35 days post-exposure ( Luxol Fast Blue 400X)
|Fig. 4: Photomicrograph showing altered architecture in cerebellar folia of 5 ppm at 14 days post-exposure (PTAH 100X)|
Similar results were reported in rat cerebellum exposed to mercury vapour (Sorensen et al., 2000) and chicks exposed to methyl mercury in ovo (Carvalho et al., 2008). At 14 days post-exposure, majority of the purkinje cells became irregular to elongate with few remained in original pyriform or flask shape; and at 35 days post-exposure, they turned into spherical or round, irregular and flatten forms.
Fig. 5: Photomicrograph showing the architecture of cerebellar folia in control at 14 days( PTAH 100X)
|Fig. 6: Photomicrograph showing altered architecture in cerebellar folia of 2.5 ppm of 14 day post- exposure (PTAH 100X)|
Likewise, Heath et al. (2010) reported various shapes due to the decreased Purkinje cell soma in MeHg treated rats. On the contrary, Hunter and Russell (1954) and Eto (1997) reported normal and well preserved Purkinje cells in MeHg poisoned Humans. The architecture of basket around the Purkinje cells formed by axons of basket cells was disrupted greatly in 5ppm group compared to 2.5 ppm group during 14 days post-exposure (Fig. 1). Similarly, Hunter and Russell (1954) observed that Purkinje cells were devoid of basket fibers in the humans exposed to industrial MeHg compounds. Dendritic arborization and reduced thickness of cytoplasmic process was noticed in both treated groups compared to control group at 35 days post-exposure. Consistently, Kim (1971) observed considerable degree of degeneration in myelin in invitro myelinated cultures of new born mouse cerebellum intoxicated with methyl mercury acetate for 18-24 days.
The cavity like vacuoles observed around the Purkinje cells were larger in the 5 ppm group compared to the 2.5 ppm group at 35 days post-exposure (Fig. 7 & 8). Further, some of the Purkinje cells showed mild vacuolation in the nucleus and cytoplasm, and a few attained pyknotic nucleus and irregular shape due to karryolysis and loss of nucleoplasm in 2.5 ppm group at 35 days post-exposure. Similar vacuolation was reported by Heath et al. (2010) in experimental rats exposed to methyl mercury at various levels. Further, some of the Purkinje cells attained irregular shape and position due to loss of nucleoplasm and karryolysis and also obtained pyknotic nucleus at 35 days post-exposure.
|Fig. 7: Photomicrograph showing cerebellar cortex in 2.5 ppm at 35 days MeHg exposure (Trichrome 400X)||Fig. 8: Photomicrograph showing cerebellar cortex in 5 ppm at 35 days post-exposure (Trichrome 400 X)|
Influence of Varying Levels of MeHg on Granular layer of Cerebellum
The influence of varying levels of MeHg on the thickness of Granular layer at 14- and 35- days post-exposure is presented in Table 4. The decreased number and density of granule cells reduced the average thickness of granular cell layer in both the exposure groups at 14 days post-exposure, which further decreased at 35 days post-exposure. Correspondingly, Roegge et al. (2004), Sakamoto et al. (2004), Howard and Mottet (1986), Clarkson and Magos (2006), and Do Nasciento et al. (2008) recorded the reduction or loss in the volume of granular cell layer in rats (exposed prenatally to 2-50 mg), mice (exposed prenatally to 0.01 to 0.4 mgs), rats (exposed to 0, 1, 3, and 5 ppm/day), rats (exposed to 12.5 ppm through drinking water), and humans, respectively. On the contrary, Saas et al. (2001) reported abnormal thickening of external granule cell layer in developing humans exposed to MeHg.
Table 4: Average thickness of granular layers in cerebellar cortex on MeHg exposure
|14 d post-exposure||35 d post-exposure||14 d post-exposure||35 d post-exposure||14 d post-exposure||35 d post-exposure|
|Control||72.8 µm||71 µm||53.4 µm||61 µm||48.2 µm||48 µm|
|2.5 ppm||62.4 µm||66.6 µm||48.2 µm||58.2 µm||45.8 µm||41.2 µm|
|5 ppm||58 µm||63 µm||46.4 µm||52.2 µm||46.2 µm||42 µm|
Shape of the granule cells were altered in both the treated groups at 35 days of exposure. The granular cells were penetrated towards molecular layer at some places in both the treated groups at 14 days post-exposure, as observed by Heath et al. (2010). Few areas of granular layer showed vacoulation due to degeneration of cerebellar glomeruli at 35 days post-exposure. Similar observations were reported by Kim (1971) in myelinated cultures of new born mouse cerebellum exposed to methyl mercuric acetate for 18-24 days. Disorientation of granular cells and alteration in cerebellar glomeruli was evident at greater levels in 5 ppm group compared to 2.5 ppm group at 14 days post-exposure. Network clusters of cerebellar glomeruli were observed at few places in both the exposure groups revealing intact glomeruli cells. In this context, Fonnum and Lock (2000) affirmed that the Purkinje cells and the granular cells are the most important targets in rats cerebellum exposed to MeHg.
Majority of the foliae in cerebellum exhibited hemorrhages with increased severity in 5 ppm group at 35 days post-exposure(Fig. 7, 8 &9).
Fig. 9: Photomicrograph showing cerebellar cortex in control at 35 days (Trichrome 400X)
Takahasi et al. (2017) and martin et al. (2000) also reported hemorrhages in cerebellum of rats exposed to methyl mercury. Concurrently, Husain et al. (2014) elucidated marked congestion of the blood capillaries with prominent perivascular fibrosis in cerebellum of adult albino rats with oral administration of mercuric chloride @ 0.33mg/kg b. wt. Furthermore, lighter cells or Golgi cell type II were observed in white matter over axons of the inner white matter. The lighter cell population was increased and the ratio of granule cells and Golgi cells appeared to be decreased in 5 ppm than in 2.5 ppm group at 35 days post-exposure. There is no report to substantiate the findings pertaining to the effect of MeHg on the lighter cell population and ratio of granule to golgi cells in the cerebellum.
Present study concluded that degeneration of granular cells, loss of Purkinje cell arborization and disruption of basket fibers around Purkinje cells may lead to disorganization of cerebellar glomerulus in granular cell layer and cerebellar circuitry of the cerebellum that in turn would affect the coordination leading to cognitive impairment in methyl mercury treated rats.
The presented manuscript is a part of the first author’s MVSc dissertation. The authors are grateful to the management of Bristol-Myers Squibb Company (BMS) Bangalore for providing the PG Fellowship Grant for animals and consumables and Department of Veterinary Anatomy, N.T.R College of Veterinary Science, Gannavaram AP India for providing infrastucure facilities for the study.