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Abstract Acute pancreatitis is a serious inflammatory disease associated with auto-digestion of the gland. This work was aimed to study the role of BM-MSCs as a therapeutic modality in acute pancreatitis induced by L-arginine in rats. It was also aimed to compare the efficacy of early versus late injection of BM-MSCs in restoration of normal exocrine pancreatic parenchyma. Sixty five adult male albino rats weighing 200- 250 gm were used in this study. In addition 10 male albino rats weighing 80-100 gm and aging 3-6 weeks were used for preparation of BM-MSCs. The experimental animals were classified into three groups: Group I (control group): this group consisted of fifteen animals. It was further subdivided into three subgroups, 5 rats each: Subgroup ІA: It included 5 male albino rats that were sacrificed after 24 hours from injection of equivalent dose of saline. Subgroup ІB: It included 5 male albino rats that were sacrificed after 72 hours from injection of equivalent dose of saline. Summary 205 Subgroup ІC: It included 5 male albino rats that were sacrificed after 2 weeks from injection of equivalent dose of saline. Group ІІ: L-arginine induced pancreatitis group: It included 30 male albino rats in which acute pancreatitis was induced. It was further subdivided into three subgroups, 10 rats each. Subgroup ІІA: It consisted of 10 rats which were allowed to live for 24 hours after the last injection of L-arginine. Subgroup ІІB: It included 10 rats which were allowed to live for 72 hours after the last injection of L-arginine Subgroup ІІC: It included 10 rats which were left for 2weeks after the last injection of L-arginine for spontaneous healing of pancreatitis. Group ІІІ: Stem cells treated group: It included 20 male albino rats in which acute pancreatitis was induced followed by injection of cultured and labeled BM-MSCs in phosphate buffer Summary 206 saline in rat tail vein. It was further subdivided into two subgroups, 10 rats each. Subgroup ІІІA: It consisted of 10 rats which were injected with bone marrow derived mesenchymal stem cells 24 hours after the last injection of L-arginine. They were allowed to live for two weeks. Subgroup ІІІB: It included 10 rats which were injected with bone marrow derived mesenchymal stem cells 72 hours after the last injection of L-arginine. They were allowed to live for two weeks. One rat from each of subgroups IIIA and IIIB was sacrificed after one week to ensure homing of BM-MSCs into rat pancreas. Blood sample were collected for laboratory serum amylase then pancreatic specimens were obtained, fixed and processed for histological study. Other small pieces of pancreatic tissues (1mm³) were freshly cut and fixed immediately in buffered formol glutaraldehyde (pH 7.3). Semithin sections were prepared and stained with toluidine blue. Summary 207 Morphometric studies were done for area percentage of collagen fibers using Maisson’s trichrome stained sections and PCNA positive cells number using immunoperoxidase stained sections for PCNA using an image analyzer. Statistical analysis was done for evaluation of the morphometric data and level of pancreatic serum amylase using SPSS.17 program. Serum amylase showed elevated level in subgroup IIA. This level was decreased in subgroup IIB and returned to near control group value in spontaneous healing group (subgroup IIC). Also the value was near to control group in both early BM-MSCs injection (subgroup IIIA) and late BM-MSCs injection (subgroup IIIB). On light microscopic examination of H&E and toluidine blue stained sections, subgroup IIA showed focal structural changes in some lobules as well as interlobular and intralobular connective tissue. The affected acinar cells showed variable structural changes. Some acinar cells were lightly stained. Others showed vacuolated cytoplasm with pyknotic nuclei. Zymogen granules depletion was obvious in some of them. Areas of intense mononuclear cellular infiltration and oedema were also noticed. The interlobular and intralobular connective tissues were relatively thickened. Some blood vessels showed Summary 208 fibrin clots with margination and pavementation of inflammatory cells. Subgroup IIB revealed more noticeable structural changes than subgroup IIA. These changes extended to include wide areas of pancreatic lobules. Intense periacinar mononuclear cellular infiltration was detected with prominent duct system within the pancreatic lobule. Interestingly, subgroup IIC revealed loss of the normal lobulation of the pancrease. Large areas of pancreatic parenchyma were occupied by fat cells with noticeable areas of fat necrosis. Some intact acini were seen in between fat tissue. Meanwhile, the remaining acini appeared distorted, vacuolated with decrease in apical secretory granules. Rounded structures of variable size were detected which might be regenerating acini. The striking feature which was observed in subgroups IIA, IIB and IIC was the preservation of islets of Langerhans. They appeared nearly similar to control group. The surrounding pancreatic acini appeared intact and comparable to the control. In stem cells treated group, subgroup IIIA, showed normal structure with tightly packed pancreatic acini and thin interlobular septa. Most acini were formed of normal acinar cells with basal basophilia and apical secretory granules and vesicular nuclei. Few acini showed hyalinized cytoplasm. Summary 209 Meanwhile, in subgroup IIIB noticeable areas of pancreatic affection were still present. There were focal areas of disorganized acini. Localized areas of mononuclear cellular infiltration were still noticed. In Maisson’s trichrome stained sections showed progressive increase of collagen fibers deposition in all subgroups of group II to be maximum in subgroup IIC. In the treated group, collagen fibers were still noticed surrounding the acini and blood vessels in both subgroups but they were more pronounced in subgroup IIIB. Despite of that, collagen fibers were apparently less than those in subgroup IIC. These results were confirmed by the statistical study. Morphometric and statistical study for area percentage of collagen fibers revealed significant increase in subgroup IIC in relation to other subgroups. In the treated group (subgroup IIIA and IIIB) there was a significant increase as compared to the control. However they showed significant decrease as compared to subgroup IIC. PCNA stained sections revealed minimal reaction in nuclei of acinar cells in subgroups IIA and IIB. However moderate reaction was noticed in both the nuclei of acinar cells and the rounded structures in subgroup IIC. Positive reaction was distinguished in most of acinar cells nuclei in subgroups Summary 210 IIIA and IIIB. Morphometric and statistical study for number of PCNA positive cells revealed significant elevation in subgroups IIIA and IIIB in comparison to other subgroups. |