The Histogenesis Of Islets In the Human Fetal Pancreas

Transcription

1 23 J Anat. Soc. India 51(1) (2002) The Histogenesis Of Islets In the Human Fetal Pancreas Gupta, V., Garg, K., Raheja, S., Choudhry, R. and Tuli, A. Department of Anatomy, Lady Hardinge Medical College, New Delhi. INDIA Abstract. Human fetal pancreas, at an optimal stage of its development is a suitable organ for transplantation after cryopreservation, in patients of insulin dependent diabetes mellitus. This study was conducted on 40 aborted human fetuses, of weeks gestation and were divided into 5 groups at an interval of 6 gestational weeks. The pancreas was sectioned and stained for light microscopy by haematoxylin and eosin, aldehyde fuchsin and Mallory s phosphotungstic acid haematoxylin. Group I fetuses (12-18 weeks) showed arborized tubules, the cells budding out from these differentiated into primitive acini and islets, both having clusters of cubical cells. At weeks scant granules were observed in the A and B cells, which coincided with the capillary invasion of the islets. The cells were evenly distributed within the islets intermixed with each other in the older fetuses of this group, no zonal arrangement of cells was seen. Well formed islets, acini and ducts were present in Group II fetuses ( weeks). The primitive stages of islets, acini and tubules were also seen. A mature islet appeared as light stained encapsulated area, some of these were showing preponderance of A cells, fetuses of more than 20 weeks showed increase in the proportion of B cells. The granules within A and B cells were densely packed in the later fetuses of this group. A considerable expansion of exocrine tissue was seen in Group III fetuses ( weeks). The connective tissue was reduced and appeared differentiated. The cells of islets were increased in number with the predominance of B cells, the density of granules in the cells was comparable to those of the 22 to 24 weeks fetuses. No significant developmental change was observed after 30 weeks of gestation. A comparison was made for the maximum density of islets in head, body and tail region of pancreas in fetuses of Group IV ( weeks) and Group V ( weeks), highest concentration of islets was seen in tail region. The study showed that acini, islets and ducts of the gland though developing from the primitive tubules are eventually independent of each other. Key words : islets, histogenesis, fetal, pancreas development. Introduction : That diabetes mellitus occurs following total pancreatectomy in the dog was serendipitously discovered by Mering and Minkowski (1890) which marked the beginning of pancreatic era This was put on a firm footing after the outstanding investigative work of Banting and Best (1922), when they prepared a pancreatic extract which was used for the treatment of diabetes mellitus. More recently clinicians are making an attempt to treat the patients of insulin dependent diabetes mellitus by transplantation of fetal pancreas. Clinical studies like these are not complete without the knowledge of developmental and morphological studies. A study of Bensley (1911) in guinea pigs reported that both acini and islets have separate origin from tubules and overlapping of islets and acinar cells sometimes gives an intermediate picture that might suggest a transformation between the two. The work of Vincent (1924) on human fetal pancreas although reported the development of islets and acini from tubules but inferred that transition from islets to acini or vice-versa can occur and developmentally they are not independent structures. Conklin (1962), Clark and Grant (1983) and Van Assche, Aerts and De Prins (1984) observed that the tubular cells developed into either the islets or acini and both are developmentally and functionally independent of each other. Bencosme (1953) however found that the mature islets develop from differentiated acini and not from the tubules. In view of these discrepancies this study was undertaken to see the origin and development of islets and its two major cells i.e. alpha and beta presently termed as A & B cells respectively, as this has relevance in fetal tissue transplants. Materials and Methods : Forty aborted human fetuses weeks gestational age with no obvious congenital abnormality were obtained from the department of Obstetrics and Gynaecology of Smt. Sucheta Kriplani hospital. These were arranged into five gestational age groups of 6 weeks interval. The fetuses of less than 12 weeks gestation could not be procured due to non availability, as these are aborted by suction and evacuation technique. The fetuses were immediately fixed in 10% formalin for 1-2 hours. In all the fetuses a cruciate abdominal incision was made for better preservation. The pancreas was then dissected and fixed in 10% formalin for hours, and processed for paraffin sections of 5m thickness.

2 24 The sections were stained for light microscopic study with haematoxylin and eosin, aldehyde fuchsin (Gomori) and Mallory s phosphotungstic acid haematoxylin. Observatins : The observations were made for each gestational group of 6 weeks interval as given below (Table I). TABLE I Distribution of fetuses in gestational groups : Group Age (weeks) No. of fetuses Group I weeks 16 Group II weeks 13 Group III weeks 7 Group IV weeks 2 Group V 36.I-40 weeeks 2 Group I (12-18 weeks) In fetuses between weeks the parenchyma consisted of mesenchymal tissue into which were embeded branched tubules with wide lumen. The tubules were lined by simple cuboidal epithelium with large and vesicular nuclei. At various places especially at the end of the tubules, budding was seen forming primitive acini and islets. The connections being in the form of cords of cells to both these entities were well defined (Fig. 1). Few groups of undiffferentiated cells were also seen. The parenchyma had begun to organize into lobes and lobules with abundant mesenchymal tissues surrounding it. Endocrine component i.e. the islets, were small and mainly spherical well defined constituents of the pancreatic parenchyma. The cells of the islets were aggregated in the center in clusters with a wide peripheral space. The mesenchymal connective tissue formed an ill defined capsule around the islet. The cytoplastmic stain (haematoxylin and eosin) showed the presence of more numbers of A cells than B cells, the former had eosinophillic and the later had basophillic cytoplasm. The characteristic granules of A and, B cells were not seen at this stage. The cells were round or oval in shape and had a dark nuclei. Few mitotic figures were also seen. Capillaries were not seen within the islets but were present in the surrounding mesenchyme. In the older fetuses of group I ( weeks), the branching was more conspicuous, lobes and lobules established. The size of the islets had increased having a distinct capsule and showed vascularisation. The cells were more evenly distributed. The A and B cells granules were seen in the 13.2 weeks embryo (Fig 2) and stained eosinophillic and basophillic with haemotoxylin and eosin and reddish orange and purple with aldehyde fuchsin respectively. The A cells were ovoid with a dense staining nuclei while B cells were more rounded, fewer with vesicular nuclei. The capillaries were seen to be forming a network within the varied size islets in the 14 weeks embryo (Fig. 3). Group II ( weeks) Parenchyma was well organized into lobes and lobules. Fewer sections of tubules were seen because of their differentiation into ducts, both interlobular and intralobular. The mesenchymal tissue was reduced due to acinar proliferation. Undifferentiated mesenchymal cells were still found in the interlobular and intralobular tissue. The islets were larger and well encapsulated and the cells were closely packed. The capillaries appeared to be more compressed (Fig. 4). The A and B cells had increased in number as well as in size. The granules appeared well established and densely arranged within the cytoplasm at this stage. The A cells were relatively more in number than the B cells but after 20 weeks an increase in the proportion of B cells was observed. Group III ( weeks) Fig 5 The intralobular connective tissue was well differentiated while the interlobular one still had some mesenchymal cells. The ducts were better formed and the connective tissue condensation was visible around the ducts though not very compact. The islets were markedly increased in number and widely distributed. Some were lying close to acini while an occasional one seen within interlobular connective tissue. The B cells were more in number and were larger than A cells. The density of granules in A and B cells appeared similar to that seen in later

3 Gupta, V. et al fetuses of previous group and localised closer to the capillaries. Group IV ( weeks) & Group V ( weeks) Fig 6 The number of fetuses in these two groups were less with no developmental peculiarity and hence the fetuses had been clubbed together. A well defined architecture of the pancreatic tissue was seen at all stages. No branching pattern was present in fetuses of more than 34 weeks, but some amount of undifferentiated mesenchymal tissue was seen. Acini and ductal system were well formed. Islets were large and prominent and were seen to be more concentrated towards the tail region, when sections at head, body and tail of pancreas were compared. Discussion : The selection of an appropriate developmental stage of fetal pancreas is of paramount importance for the successful transplant of pancreas in patients of insulin dependent diabetes mellitus. The earliest fetus procured in the study was of 12 weeks gestation. It s parenchyma appeared as collection of branched tubules lined by cuboidal cells. Groups of cells from these proliferated to form primitive acini, islets and ducts. A similar architecture was reported by, Conklin (1962) and Like and Orci (1971) in 10 weeks old fetuses. This suggests that in early weeks of fetal maturation primarily there is budding of cells from the primitive tubules with little or no differentiation into acini, islets or ducts. Organization of parenchyma into lobes and lobules had begun at 12 weeks, but was well defined in fetuses of weeks, a feature, same as that reported by Conklin (1962) at 13 weeks and Clark and Grant (1983) in fetuses of 12 weeks. Contrary to above Bjorkman, Hellerstorm, Hellman and Peterson (1966) observed an organized lobular pattern at 17 weeks. The primitive islets budding out from tubules, evident in the youngest fetus (12 weeks) of the present series is in conformity with the work by Robb (1961) and Achaya and Anand (1965). While Conklin (1962), Clark and Grant (1983) and Van Assche et al (1984) found them at an earlier stage. These were seen connected to tubules. The connection to the tubules were severed, and the relation was infrequently observed after 20 weeks of gestation. This suggests the origin of islets from the tubules and the subsequent establishment of their independent identity. Bencosme (1953) to the contrary reported the development of islets from diferentiated acini, while Vincent (1924) described the islet as temporarily modified portions of zymogenous tissue. However Conklin (1962), Like and Orci (1971), Clark and Grant (1983) and Van Assche et al (1984) observed similar developmental pattern as that of present study. The first islet cell type to appear was A cell as observed by Conklin (1962), Like and Orci (1971) and Van Assche et al (1984) at 8 to 9 weeks. The appearance of first islet cell could not be identified in this work due to non availability of fetuses of less than 12 weeks. The presence of two types of cells were seen within the islet having acidophilic and basophilic cytoplasm in the 12 week fetus of the present work and were termed as A and B cells respectively. The granules in them were identified at weeks with haematoxylin, eosin and special stains. The A and B cells of islets were also reported by Robb (1961) at 12 weeks. A third type of cell D cell was described by Conklin (1962) at 13 weeks and Like and Orci (1971) at 11 weeks, while Van Assche et al (1984) using immunocytochemical techniques have identified 5 types of cells. (A, B, D, D1 & HPP cells). A peculiar stage in the development of islets is the mantle islet stage as reported by Robb (1961), where a zonal arrangement of cells was seen with B cells in the center, and A cells at the periphery. This was also observed by Conklin (1962) in fetuses up to the age of weeks. The mantle stage was not noticed in the present work, instead the A and B Cells were seen intermixed with each other throughout the development. This stage is an adult characteristic of animals like rat and rabbit and not seen in adult human pancreas, this difference in arrangement of cells probably makes man prone to diabetes, (Achaya and Anand, 1965). Structural organization of cells of islet in different species has a functional significance as evidenced by Elayat, et al (1995). The cell buds destined to form islets were seen enclosing a group of capillaries at weeks, this coincided with the appearance of granules in them, thus indicating the endocrine nature of 25

4 26 the gland. Conklin (1962) reported the capillary invasion of islets at 12 weeks. A capsule of loose connective tissue was seen surrounding the developing islets, separating them from the acinar tissue, however, few islets were lying in close contact with the acini. Henderson, Daniel and Fraser (1981) in rat pancreas have shown that endocrine pancreas plays a significant role in metabolic activity of exocrine but whether the close association of acini and islets plays any significant role needs to be investigated. In the present investigation on the regional distribution of islets in the pancrease of weeks fetuses it was seen that the tail region has significantly high density of islets as compared to head and body. This was supported by Elayat et al (1995) in rats. The study by Saito, Iwama and Takahashi (1978) on human adult pancreas reported highest volume density of islets in tail region. The significance of difference in regional distribution of islets is not clear, however, it may be reflecting functional or anatomical adaptation. The human infant and fetal pancreas are a potential source of islet tissue for transplantation, (Sutherland et al, 1976 and Sandler et al, 1982 respectively). Sandler et al (1982) further reported a positive correlation between the pancreatic insulin content and crown rump length. Dense aggregations of granules were seen in A and B cells of week fetuses of present series. This may have association with the amount of insulin secreted by them. However, it is crucial to identify an appropriate stage of fetal pancreas development, that will be an optimal transplant material. Brown et al (1980) in a comparative study between human and rat fetal pancreas reported that human fetal pancrease of weeks may be a suitable donor material but a detailed bio-chemical and ultrastructural analysis is required for a more accurate stage. Thus the success of pancreatic transplant requires the knowledge of it s development, morphology, insulin content and it s response to glucose at various stages of islet genesis making this study significant. 2. Banting, F.G. and Best, C.H. (1922): The internal secretion of pancreas. Journal of Laboratory Medicine, 75: Bencosme, S.A. (1953): The Histogenesis and cytology of the pancreatic islets in the rabbit. American Journal of Anatomy, 96: Benseley, R.R. (1911): Studies on the pancreas of guinea pig. American Journal of Anatomy, 12: Bjorkman, N.; Hellerstrom, C.; Hellman, B and Peterson, B. (1966): The cell types in the endocrine pancreas of the human fetus, Zeitschrist Fur Zellforschung, 72: Brown, J.; Kemp. J.A.; Hurt, S. and Clark, R. (1980): Cryopreservation of human fetal pancrease. Diabetes. 29(1): Clark, A. and Grant, A.M. (1983): Qunatitative morphology of endocrine cells in human fetal pancreas. Diabetologia, 25: Conklin, J.A.(1962): Cytogenesis of human fetal pancreas. American Journal of Anatomy, 111: Elayat, A.; Naggar, E.L.; Mostafa, M. anmd Tahir, M. (1995): An immunocytochemical and morphometric study of the rat pancreatic islets. Journal of Anatomy, 186: Henderson, J.R.; Daniel, P.M. and Fraser, P.A. (1981): The pancreas as a single organ: The influence of the endocrine part of the gland. Gut, 22: Like, A.A. and Orci, L. (1971): Embryogenesis of the human pancreatic islets ; A light and electron microscopic study. Diabetes, 21(2): Mering, V. and Minkowsky (1890): Arch. f. Experium. Patti.U.Pharm. Bd 26: 371. cited by Bensley, R.R. (1911). 13. Robb, P. (1961): The Development of islets of Langerhans in the human fetus. Quarterly Journal of Experimental Physiology, 46: Saito, K.; Iwama N and Takahashi, T. (1978): Morphometrical analysis on topographical difference in size distribution, number and volume of islets in human pancrease. Tohoku Journal of Experimental medicine 124: Sandler, S.; Anderson, A.; Hellerstrom, C.; Peterson, B.; Sweene, I.; Bjorken, C. and Groth, C.G. (1982). Preservation of morphology, insulin biosynthesis, and insulin release of cryo preserved human fetal pancreas. Diabetes, 31: Sutherland, D.E.R.; Matas, A.J.; Steffes, M.W. and Najarian, J.S. (1976): Infant human pancreas: A potential source of islet tissue for transplantation. Diabetes, 25(12): Van Assche, F.A., Aerts, L. and De Prins, F.A. (1984): The fetal endocrine pancreas. European Journal of Obstetric and Gynaecology Reproductive Biology 18: Vincent, S. (1924): The relationship between the islets of Langerhans and the zymogenous tissue of the pancreas. Lancet, 206: This Article Can be Downloaded / Printed Free from References : 1. Achaya, S. and Anand, C. (1965) : Histogenesis of Pancreatic islets in the human embryo. Journal of Anatomical Society of India, 14(2):

5 Opp. 24 Fig. 1 Photomicrograph of pancreas at 12 weeks showing differentiation of tubular buds (t) into acini (a) and islets (thin arrow). Cords of cells (c) connecting these to the tubules also seen. H&E x 100. Fig. 2. Photomicrograph of islet of Langerhans of 13.2 weeks showing the presence of cytoplasmic granules in A or alpha cells (thick arrow) and B or beta cells (thin arrow). H&E x Fig. 3. Photomicrograph at 14 weeks showing lobules (l), islets of variable sizes (thin arrow) and the capillary (cap) network. H&E x 100.

6 Opp. 25 Fig. 4. Photomicrograph of islet at 24 weeks showing densely packed A (thick arrow) and B (thin arrow) cells around the capillaries (cap). Aldehyde Fuchsin x Fig. 5. Photomicrograph at 26 weeks showing typical lobulated pattern with differentiated intralobular connective tissue, condensation of mesenchymal tissue around the interlobular duct (thick arrow), and an islet (thin arrow) lying within the interlobular tissue. H&E x 100. Fig. 6. Photomicrograph at 37 weeks showing compact multi-layered musculature around the duct (thick arrow) well formed acini (a) and islets (thin arrow). Mallory s phosphotungstic acid haematoxylin x 400.