Ultrastructural Architecture Studies of Pancreas in Guinea pig

The guinea pig (Cavia porcellus) belongs to the rodent order but differs in many metabolic aspects from rat and mouse and, in particular, exhibits several unique features to humans in its lipid metabolism. Guinea pigs played a major role in the formation of germ theory through the experiments of Louis Pasteur, Emile Roux and Rober Koch (Buchholz and Schoeller, 2004). Guinea pigs had an unusual insulin mutation and the suitable species for the anti-insulin antibodies production (Chan et al., 1984). The pancreas is an accessory organ of the digestive system and also an important endocrine organ of vertebrates. Its function is to regulate blood sugar levels by secretion of hormones like insulin, glucagon, stomatostatin and pancreatic polypeptide. As an exocrine gland, it produces pancreatic juice for digestion (Sisson et al., 1975). To evaluate the pathologic and clinical reactions, it is essential to understand the normal structure of the organ. Hence, a thorough knowledge on the SEM and TEM of the pancreas is necessary for the proper diagnosis and treatment of ailments. The experimental animals like guinea pigs, mouse, rats, pigs and monkeys play an important role in human medicine, since the anatomical structure of the organs of experimental animals is similar to that of  humans (Al-Sharoot, 2014). Studies through the SEM, the smallest acinus observed measured about 8µm in diameter and looked like a bud. The acinar cells were round or oval in shape and measured 30-40µm in diameter in human pancreas. Most acini, were composed of crooked and branching cords of acinar cells (Takahashi, 1984). In guinea pigs, at two days of post-natal life, the acinar cell of the pancreas was found to be small, with the large nucleus, Golgi complex, cisternal Rough Endoplasmic Reticulum (RER), mitochondria and small zymogen granules (De-Assism et al., 2003). Fattah (2008) stated that the presence of electron dense granules in beta cells of albino rat. Blood flow pattern in exocrine and islet part of pancreas of human was studied by El-Gohary and Gittes (2018). Longnecker (2021) stated that the centroacinar cells in humans can be differentiated from the absence of zymogen granules and less endoplasmic reticulum.
       
Due to paucity of literature available in guinea pigs, the present basic research on the scanning and transmission electron microscopic studies of pancreas was carried out.

Sample collection
 
The present study was conducted at the Department of Veterinary Anatomy, Madras Veterinary College, Chennai during the period from 2019 to 2021. Six adult healthy guinea pigs of 16-32 weeks of age (Irrespective of sex) were procured from the Department of Laboratory Animal Medicine, Madhavaram Milk Colony, TANUVAS, Chennai-51. After collection of the guinea pigs, they were euthanized as per the standard operating procedure by using the Carbon dioxide asphyxiations as per CPCSEA norms and they were subjected for the dissection. After careful dissection of the animals, pancreas was dissected out.
 
Scanning electron microscopy
 
The sections of pancreas of adult guinea pigs were pre-fixed in 2.5 per cent glutaraldehyde-PBS at 4°C for 2 hours. Then samples were washed in PBS and post-fixed in 2 per cent Osmium tetroxide-PBS for 2 hours. Then samples were washed and dehydrated through graded series of ethanol. The samples were mounted on the stubs and coated with gold by a sputter coater as stated by Karahan et al., (2007). The SEM images were observed by a Phenom Pro scanning electron microscope at CATERS facility in Central Leather Research Institute, Chennai.
 
Transmission electron microscopy
 
Pancreas samples of adult guinea pigs were collected and prefixed at three per cent glutaraldehyde and stored at 4°C. Subsequently, the tissues were washed, three changes (each 30 minutes) in cold sodium cacodylate buffer solution (pH. 7.4) and post fixed in one percent osmium tetroxide for two hours at 4°C. The tissues were then dehydrated in ascending grades of alcohol, propylene oxide: epoxy resin mixture and embedded in Epon-araldite mixture. Semithin (one micron) sections were stained by toluidine blue. Ultrathin sections (600 Å to 900 Å) were prepared on Leica ultracut microtome, mounted on uncoated copper grids and stained with saturated solution of Uranyl acetate and lead citrate. The ultra thin sections were examined under Jeol 1400 Transmission Electron microscope, computer augmented transmission Electron microscope operated at 60-Kilowatt ampere (KV) (Rajathi et al., 2022).
 
Ethics statement
 
The animal ethical committee of the Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India had approved the collection of laboratory animals and handling as per the Ethical Committee approval (Lr. No. 1467/DFAB/IAEC/2018 dated 13.07.2018). The methods were performed in accordance with the guidelines of the institutional ethical committee of TANUVAS, India. All procedures were performed in accordance with the CPCSEA norms (Periera et al., 2004).

The scanning and transmission electron microscopic architecture of pancreas in guinea pigs were similar to other mammals except the following unique findings. Ultrastructurally, guinea pig pancreas showed apparent structural features of digestion and blood sugar maintenance.
 
Scanning electron microscopy
 
In the present study, the pancreatic parenchyma was covered by the dense irregular connective tissue capsule with different orientation of connective tissue fibres. The connective tissue fibres were found between the lobules and also found between the individual acini of 16 week-old guinea pig pancreas (Fig 1). It was compound alveolar gland. Each lobule contained many acini which were connected to each other by a thin, long duct with branched pattern arrangement. Takahashi (1984) mentioned about the shape of the acinar cells as round or oval in shape which was similar to the observations of the present study. The acinar cells were connected and drained by intercalated duct which was thin and small. The pancreatic lobule was drained by interlobular duct which was thicker than intercalated duct and the pancreatic lobe was drained by interlobar duct which was larger in diameter than interlobular duct. In between the acini and between the lobules, large fat lobules were also found. Interdigitations of nearby acinar cell membranes were also found. Large blood vessels supplying the lobes and lobules of pancreas were found in the low SEM magnification.
 

 
Transmission electron microscopy
 
The pancreatic tissue consisted of glandular lobules comprised of acini, islets of Langerhans and connective tissue between the lobules. Inside the lobules, lot of small blood vessels and blood capillaries were found surrounding the gland which contained few telocytes. Similar identification of cells was observed in pancreas of aquatic turtles as per (Gandahi et al., 2020). The pancreatic acinar cells were pyramidal in shape and had large irregular shaped euchromatin nucleus with areas of electron dense particles at the periphery. Nucleolus was clear, electron dense and occupied an eccentric position within the nucleus. The cytoplasm was fully packed with cisternae of rough endoplasmic reticulum. The apical portion of the cytoplasm of the acinar cells had numerous homogenous electron dense bodies named zymogen granules. Numerous mitochondria and golgi complexes were also present in the acinar cell cytoplasm along with the zymogen granules and rough endoplasmic reticulum of 20 week-old guinea pig pancreas (Fig 2). The acinar lumen had electron dense particles. Similar observations were also recorded in guinea pigs by De-Assis et al., (2003) and in camel by Hafez and (Zaghloul, 2017).
 

       
Some acinar cells were electron dense and few acinar cells were electron lucent. The electron dense acinar cells had more proportion of rough endoplasmic reticulum than electron lucent cells. These acinar cells were connected to adjacent acinar cells by junctional complexes. Similar observations were also reported by Beheirya et al., (2018) in goose pancreas. The apical portion of the acinar cells showed numerous microvilli of 20 week-old guinea pig (Fig 3). The 5-6 acinar cells were found surrounding the centroacinar cells which was found smaller in size when compared to the acinar cells. The centroacinar cells had many electron dense particles in the cytoplasm. The cytoplasmic and nuclear contents are not clearly distinguishable in the present study. Many microvilli were found surrounding the cell membrane of the centroacinar cell of pancreas of 20 week-old guinea pig (Fig 4). In the proximity to the blood capillary, a special type of interstitial cell named telocytes were found with many telopodias in the exocrine part of pancreatic parenchyma which had association with the acinar cell and blood vessel of pancreas of 20 week-old guinea pig (Fig 5). Nicolescu and Popescu (2012) reported that telocytes acted as important players in intercellular signalling via stromal synapses and shed vehicle transfer in human pancreas. Similar observation regarding the presence of telocytes in the stroma of pancreas was also observed in human and turtle pancreas by Nicolescu and Popescu (2012) and Gandahi et al., (2020).
 

 

 

       
Among the four islet cell types, alpha and beta cells could be identified in the present study. Alpha cells had irregular outline and had numerous homogenous small membrane bound granules with moderate to high electron density. Similar observations were also recorded by Lacy and St-Louis (1957) in guinea pig pancreas and by Hafez and Zaghoul (2017) in camel pancreas. Mostly the granules were positioned towards the periphery of the cytoplasm. The alpha cells were observed with ovoid nucleus with indentation, with electron dense condensed chromatin particles located at the periphery as stated by Lacy and St-Louis (1957) in guinea pig. The cytoplasm of alpha cells was found with various sized vacuoles and rough endoplasmic reticulum. Smooth and rough endoplasmic reticulum also found in the cytoplasm of alpha cells. Golgi complex also found in the cytoplasm of alpha cells as vesicles of pancreas of 20 week-old guinea pig (Fig 6).
 

       
Beta cells were distributed throughout the islet both in the center and periphery. Beta cells were almost similar in size to that of alpha cells. But Hafez and Zaghloul (2017) found large beta cells when compared to alpha cells. The cytoplasmic granules were fewer in number than alpha cells as per the results of Lacy and St-Louis (1957) in the pancreas of dogs, rabbit and guinea pig. The granules were spherical in shape, larger in size and distributed throughout the cytoplasm whereas in rabbits, the granules were rod shaped (Lacy and St-Louis, 1957) and in dogs, the granules were irregular in shape and small, round and indistinct in rabbit pancreas and round in rats (Lacy and St-Louis, 1957). The appearance of electron dense granules was also recorded in albino rat (Fattah, 2008). The beta granules were membrane bound with an electron lucent halo around the granules. Islets were vascularized and endothelial lining of the blood vessels were fenestrated of pancreas of 20 week-old guinea pig (Fig 6).

The scanning and transmission electron microscopic architecture of pancreas in guinea pigs were similar to other mammals except the following unique findings. Ultrastructurally, guinea pig liver showed apparent morphological features of carbohydrate, protein and fat digestion and blood sugar maintenance. The pancreatic parenchyma was covered by the dense irregular connective tissue capsule with different orientation of connective tissue fibres. The connective tissue fibres were found between the lobes and lobules and also found between the individual acini. Each lobule contained many acini which were connected to each other by a thin, long duct with branched pattern arrangement. The acinar cells were drained by intercalated duct, interlobular duct, interlobar duct and interlobular duct were found with increasing diameter in the low SEM magnification. The pancreatic acinar cells were pyramidal in shape and had large irregular shaped euchromatin nucleus with areas of electron dense particles at the periphery. Nucleolus was clear, electron dense. The cytoplasm was fully packed with cisternae of rough endoplasmic reticulum. The apical portion had numerous homogenous electron dense zymogen granules. The electron dense acinar cells had more proportion of rough endoplasmic reticulum than electron lucent cells. The apical portion of the acinar cells showed numerous microvilli. Alpha cells had irregular outline and had numerous homogenous small membrane bound granules with moderate to high electron density. Beta cells were almost similar in size to that of alpha cells. The cytoplasmic granules were fewer in number than alpha cells. The granules were spherical in shape, larger in size and distributed throughout the cytoplasm. The beta granules were membrane bound with an electron lucent halo around the granules.

The authors are grateful to Dean, Madras Veterinary College, Chennai for providing facilities for research.

Data available on request from the corresponding author.

The authors declare no potential conflict of interest.