Introduction

Mongooses are small carnivores occupy- ing various regions from Africa to southeast Asia. The genus Herpestes contains 10 spe- cies and is considered the oldest genus in the order Carnivora, dating back to approx- imately 30 million years (Shil et al., 2012). The Indian gray mongoose (Herpestes ed- wardsii) is a mongoose species that is prin- cipally found in southern Asia particularly India, Pakistan, the South of Iran, Sri Lanka and some other parts of Asia. Also, body mass in males is considerably more than fe- males (Rasouli et al., 2015).

The brain is made of three main parts: ce- rebral, cerebellum, and medulla oblongata. The cerebrum is the largest part of the brain and involves reasoning, learning, sensory perception, and emotional  responses.  On the other hand, the cerebellum is responsi- ble for the maintenance of equilibrium of the body by coordinating somatic motor activity and by regulating muscle tone. This part is more important in active vertebrate genera since it is intimately involved in the control and maintenance of muscle tones and there- by motor coordination (Dyce et al., 2017). Brain study in animals is important for ani- mal’s welfare and knowledge about the ner- vous system function, physiology, behavior habits, and developmental anatomy.

Histological studies on cerebrum and cer- ebellum have been of interest to the anatom- ical scientists in the last decade. The micro- anatomy of the cerebrum has been studied and described in human, mouse (Treuting and Dintzis, 2012), rabbit, rat (Ibegbu et al., 2014), and guinea pig (Musa et al., 2016). Moreover, (Pal et al., 2003; Irimescu et al., 2015; Treuting and Dintzis, 2012; Danmai- goro et al., 2016; Sur et al., 2011; Musa et al., 2016; Beheiry, 2015) provide valuable

information about the cerebellum histolog- ical aspects in the fowl, chinchilla, mouse, catfish, birds, guinea pig, and camel, respec- tively. Also, Jardim-Messeder et al. (2017) carried out extensive comparative studies on the number of brain neurons and their brain- size in various carnivores including one of the mongoose species.

Comparative morphology has been served as evidence for evolution and indicates that various animals share a common ancestor. This allows scientists to classify animals based on similar characteristics or diversity of their body structures. There is a dearth of information on the comparative histological organization of the cerebrum and cerebel- lum in the carnivores. In addition, this study may be helpful for a better understanding  of the brain structural variations related to behavioral habitats and functional anatomy of this species.

Our study aimed to enrich the current data pool by providing a comparative histological description of the mongoose’s cerebrum and cerebellum.

Materials and methods

Eight adult mongooses (4 male and 4 female) which have been found in the sur- rounding areas of Shiraz (capital of the Fars province-Iran) were used for this study over the last two years. The age of the animals was estimated to be more than one-year-old with respect to the teeth inspection. These fresh specimens in an end-stage disease or the status of approaching death (severe hy- pothermia; 36 ○C body temperature) were ad- mitted to anatomy laboratory in veterinary faculty, Shiraz University, Shiraz, Iran. All these activities were done in coordination with ethical rules of the department of en-

vironmentt of Fars province. The obtained Licence No was: 5599/700 / 93 / p.

The animals were euthanized with Ket- amine 10% and Xylazine 2%. Then 10% buffered formalin solution was injected into the lateral ventricles by creating a small pore in the frontal bone. In this way, the fixative material was well distributed in the cerebral ventricles and the subarachnoid space and the nervous tissue became more firm. These methods were performed based on the study of Musa et al. (2016).

The specimens were then transferred into the big containers of phosphate buff- ered formalin solution, 24 h later, the skull was removed and the brain was ex- posed. Then, the tissue samples were collected from parietal and frontal lobes of the right cerebral hemisphere and right cerebellar hemisphere (Figure 1).

These samples were embedded in paraffin, cut into serial sections, and stained using a standard Hematoxylin and Eosin protocol (H&E stain). In this study, the thickness of the white matter and cortex layers in the cerebrum and cerebellum, the number of neurons and neuroglia cells per unit area, and morphological features of  the  tissue  of the organs were measured  and  record- ed by Image Pro 7 plus software (Pal  et  al., 2003). In the following, a comparison  is made between the results regarding the histological aspects of cerebrum and cer- ebellum and those of literature data about the other species.

Statistcal analysis

Analysis of morphometric data was car- ried out with Student’s t-test using the SPSS software (Version. 23) at the significance level P ≤ 0.05.

Figure 1. A: Structure of the brain after removing the skull in an adult mongoose (Dorsal view). B: Areas for tissue sampling of the cerebrum and cerebellum

Results

The mongoose’s cerebral histological section consists of two distinguished ma- terials: gray matter and white matter. The gray matter or cerebral cortex is situated externally while the white matter is situat- ed internally (Figure 2). The results of the studies on thickness measurements of the white and gray matter layers, as well as

the ratio between them in the frontal and parietal lobes, are summarized in Table 1. Further, the results of studies on the cel- lular density of neurons per unit area of gray matter, and also the neuroglia cells per unit surface area of white matter are reflected in Table 1. All the results and significant differences were performed considering P ≤ 0.05.

Figure 2. Photomicrograph of the frontal lobe of the cerebrum in the male mon- goose. Stain: H&E. White matter (wm) and Grey matter (gm)

Table 1. Thickness of the gray matter and white matter of the cerebrum, their ratio, and cellular density per unit area (Mean ± SD) both in male and female mongoose.

(* significant difference P ≤ 0.05).

The observations of the  present study showed that the layers of the frontal lobe in cerebrum cortex are  relatively  recognizable so that the molecular layer of the cortex was  a marked layer with very few cells. The out- er granular and pyramid layers are close and distinguishable from each other. The internal granular and pyramidal layers are highly in- terconnected. However, as the outside moves

to the medullary area, the pyramidal cells (Betz cells) becomes larger. The polymorphic layer also has high cell density and cellular forms. The boundary between the gray mat- ter and the white matter is also clearly evident (Figure 3A). The mentioned features are also seen in the parietal lobe. The difference is that six layers appear more clear and distinct from each other (Figure 3B).

Figure 3. Photomicrograph of cortex  layers  in  the  parietal  lobe  (A)  and  fron- tal lobe (B) in the mongoose’s cerebrum. Stain: H&E.

White matter (wm), Betz cell (red arrow), Molecular layer (1), External granular layer  (2),  External py- ramidal layer (3), Internal granular layer (4), Internal pyramidal layer (5), Polymorphic layer (6).

Histologically, the cerebellum also has white and gray matter. The cerebellar white matter was observed to be featureless. Moreover, its function as a germinal zone is controversial. Microscopic examination of  the mongoose

cerebellum samples represented the usual structure of the cerebellar cortex or grey matter in all animals: the outer, molecular, the middle layers are composed of a single row of purkin- je cells and an inner granular layer (Figure 4).

Figure 4. Photomicrograph of mongoose’s cerebellum. Stain: H&E.

White matter (wm), Molecular layer (ml), Purkinje cells layer (pl), Granular layer (gl)

The results of the thickness measurements of white matter, gray matter, and also various lay- ers of gray matter are summarized in Table 2, considering P ≤ 0.05. Further, the results from studies on density measurements of cells per

unit area (n/mm2) for different layers of gray matter are presented in Table 2. It should be noted that because of the low thickness of the purkinje cell layer, the density of these cells has been measured in unit length (n/mm).

Table 2. Thickness of the cerebellum layers and their cellular densi- ty per unit area (Mean ± SD) in the male and female mongoose

(* significant difference P ≤ 0.05).

In the molecular layer, we are able to see the stellate and basket cells, as well as numer- ous rich dendritic branches from the purkinje cells, oriented eccentrically towards the ex- ternal surface of the cortex. In this layer, the basket cells are placed next to the Purkinje cells and the stellate near the outer surface. The middle layer includes the characteristic purkinje cells. Our  photomicrographs indi-

cate that their soma is circular or oval-shaped, the length is about 20 μm, and width of about 15 μm, in both sexes. The granular layer pre- sented a high concentration of heterochro- matin granular cells and few golgi cells with a pale appearance in between. Among the granular cells, there were small spaces called cerebellar islands appearing as irregular light areas (Figure 5).

Figure 5. Photomicrograph of cortex layers of the mongoose’s cerebellum. Stain: H&E.

Granular cells of the granular layer  (a),  Golgi  cells  of the  granular  layer  (b),  Dendritic  branches  of Purkinje  cells (c), Basket cells of the molecular layer (d), Stellate cells of the molecular layer (d), Soma of Purkinje cell (f)

Discussion

The present study showed that the gray matter and white matter in the cerebrum are placed in the cortex and center, respectively, which is similar to other animals (Dellman and Eurell, 1998). In the molecular  layer, cell density was very low, due to the pres- ence of dendritic fibers of the neurons in the underlying layers, which helps to distinguish this layer from other layers (Figure 3). This layer is thicker in Indian guinea pigs than that of laboratory mouse and rats (Treuting and Dintzis, 2012). In addition, the highest proportion of gray matter to white matter was found in the frontal lobe of male mon- goose. This proportion is lower in the female mongooses in two lobes unclearly (Table 1). Under it, external granular cells are ob- served and below it, there is a row of small to medium pyramidal cells that are related to the outer pyramidal layer. In the frontal lobe, the inner granular layer is combined with the inner pyramidal layer of the large pyramidal cells and the polymorphic layer was thick with high cell density. This is while these two layers in the parietal lobe are separated and the thickness of the  polymorphic layer is less (Figure 3). Studies on guinea pigs showed that there is no external pyramidal layer in this animal, but the rest of the layers exist (Musa et al., 2016). Also, in the labora- tory mouse, the second and third layers are combined, but other layers are separated. In humans, all six layers are distinct and clear

(Treuting and Dintzis, 2012).

The remarkable point in these  layers  is the high thickness of the polymorphic lay-  er and the increased betz cells in the cortex, as compared to studies on rats, guinea pigs and rabbits (Ibegbu et al., 2014). This may be the reason why carnivores like mongoose have more sense of understanding and intel-

ligence than rodents (Kelly and Stick, 2003). Therefore, the agility and intelligence of this animal in nature are related to these charac- teristics.

In this study, it was determined that all lay- ers of the cerebellar cortex, as well as white matter in male, are more than female. How- ever, the difference is not significant (Table 2). It is necessary to note that molecular and granular layers in the cerebellar folia sum- mits are thicker and have a lower thickness in the folia fissures. This is while the white matter thickness is constant in all areas of  the cerebellum (Figure 4). However, studies on human, camel, chicken, and pigeon show that in the cerebellum folia fissure only the granular layer has a lower thickness (Pal et al., 2003; Sur et al., 2011; Beheiry, 2015).

In cellular density measurements of the ce- rebral tissue, it was found that the cell densi- ty of the neurons (n/mm2) in the female mon- goose is higher than male, while the density of neuroglia cells was higher in males (Ta- ble 2). These results are in good agreement with studies conducted on human (Witelson et al., 1995). The performed research shows that the density of cortical neurons in banded mongoose is lower than that of other carni- vores and is not related to the domestication or body mass (Jardim-Messeder et al., 2017). Given the smaller size of females, these find- ings are consistent with the present study.

Pearson (1972) stated that throughout all vertebrate genera the organization of cells and axons within the cerebellum had a sim- ilar and distinctive appearance. A number of small star-shaped cells found in a scattered manner close to the periphery of the cerebel- lar molecular layer represented stellate cells (Figure 5). This is also in agreement with studies performed on fowl, camel, catfish, and chinchilla (Irimescu et al., 2015; Dan-