Toxicology Letters, 62 (1992) 263-214 0
263
1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/$05.00
TOXLET 02771
Endocrine pancreas in the postnatal offspring of alloxan diabetic rats
Mohamed S. Abdel-Rahman”, Fakhry A. Iskanderb “Department of Pharmacology/Toxicology,
Fatma I. Elrakhawyb and UMDNJ, New Jersey Medical School, Newark, NJ (USA)
and
bAnatomy Department, Medical School, Ain Shams University, Cairo (Egypt)
(Received 13 April 1992) (Accepted 12 June 1992) Key words: Diabetic; Postnatal; Pancreatic; Rat
SUMMARY This study was conducted to investigate the morphological changes in the postnatal pancreatic islets in offspring of diabetic rats. Diabetes was induced in female Sprague Dawley rats by intravenous injection of alloxan (40 mg/kg). After 1 week, rats with blood sugar above 270 mg% were bred and watched until spontaneous delivery occurred. Litters in the following age groups were sacrificed by decapitation, 0,8, 24, 72 and 168 h, and compared with their controls. Blood sugar levels were significantly higher in the neonates of diabetic mothers immediately after delivery compared to the control, then became normal after 8 h. Islets of the offspring of the diabetics at birth showed weak positive staining for insulin using immunocytochemical techniques. By 72 h some cells showed immunopositive staining similar to the control, while at 168 h all the p (B) cells were stained normally. /l cells of the islets from the diabetic series at birth were almost completely degranulated except for scattered granules toward the periphery. Their cytoplasm exhibited glycogen and lipid accumulations. Cells also showed signs of hyperfunction in the form of an extensive endoplasmic reticulum and well-developed Golgi complex with distended Golgi cistemae. At 8 h postnatally the population of pale secretory granules was markedly increased. The changes described at birth persisted at 24 h and, to a lesser extent, 72 h after delivery. At the age of 1 week, the /? cellsappearedto be normal.
INTRODUCTION
Pregnancy in a diabetic patient resulting in infant survival during birth was one of the great medical accomplishments of the twentieth century. Prior to the use of exogenous insulin, pregnancy in a diabetic patient was virtually unheard of, or resulted Correspondence to: M.S. Abdel-Rahman, Avenue, Newark, NJ 07103, USA.
Department of Pharmacology/Toxicology,
185 South Orange
264
in severe compromise of the patient and almost certain death of the fetus or newborn. Macrosomia, fetal distress, increased morbidity and mortality were the major complications in the outcome of diabetic pregnancies [l]. Since the discovery of the diabetogenic effect of alloxan and its specific destructive effect to the B cells of the islets, animal models have been widely used to study the effect of diabetes on pregnancy [2-51. However, decreased fertility and increased mortality of the diabetic animals complicated this approach [3,6]. Prolongation of the gestational period by 2 days was recorded [5,7]. Although macrosomia was observed in the human, conflicting reports on the fetal body weights in offspring of diabetic rats have been reported. Hultquist, Angerwall, Solomon and Lazarow et al. reported increased fetal body weight of the offspring [6,8-lo]. However, Kim et al., Lawrence and Contopoulos and Eriksson et al. reported a decrease in the rat offspring body weight [5,11,7]. In the meantime, no change in the weight of the young was reported by other investigators [12]. The insulin and somatostatin concentrations of the fetal islets of diabetic rats were sigificantly lower than the control but the glucagon level was unchanged [13,7]. A positive correlation existed between the blood sugar level of the diabetic mothers and their fetuses for a few hours after the delivery [14]. Human studies revealed that the islets of Langerhans in infants of diabetic women exhibited several morphological changes such as vacuolation of the B cells [ 151.In the meantime, an increase of the islet cell volume and marked eosinophilic infiltration of the islets were shown [16-181. The morphological changes occurring in the islets of prenatal young of diabetic rats were similar to those occurring in the adult rats subjected to hyperglycemic stress in the form of degranulation and hydropic degeneration (vacuolation of the cytoplasm) of the B cells [4]. Mild hypertrophy and hyperplasia of the islets of the newborn young of diabetic rats have also been observed [8]. Aerts and Van Assche described marked degranulation, glycogen infiltration and hyperfunction of the B cells in neonates of rats made diabetic on their first day of pregnancy [19]. These rats were treated with streptozotocin (specific B-cytotoxic drug). However, the injection of B-cytotoxic drugs has been declared to have injurious effects on the conceptus or the placenta, therefore, in an animal model an interval should elapse between the administration of the cytotoxic drug and the occurrence of pregnancy [20,7]. This study was conducted to investigate the morphological changes of the islets in offspring of alloxan diabetic rats during the first week after delivery using both light and electron microscopy. The islets cells of the young of diabetic mothers were compared with those of the neonates of control animals by using the highly specific immunocytochemical technique. In addition, attention was focused on the possible occurrence of macrosomia amongst the offspring of the diabetic rats. MATERIALS
AND METHODS
Female Sprague Dawley rats, weighing 180-255 g, were purchased from Taconic
265
Farms, Germantown, NY, and quarantined for 1 week. The rats were maintained three/cage in a temperature (25°C) and humidity (50%) controlled environment with a 12-h light/dark cycle. Laboratory rodent chow (Ralston Purina, St. Louis, MO) and water were available ad libitum. Rats were fasted for 24 h before the treatment. Twenty-six rats were injected intravenously in the tail vein with alloxan monohydrate (Sigma, St. Louis, MO), 40 mg/kg body wt. Alloxan was prepared fresh for each animal as a 5% aqueous solution. Six rats were used as controls. Animals were allowed free access to food and water immediately after treatment. Blood samples were withdrawn by cardiac puncture before treatment to determine the blood sugar using glucose oxidase [21]. Four days after treatment, non-fasting blood sugar levels were measured and rats with a blood sugar level of 260 mg% or more were used for the study. Induction and the course of pregnancy One week after treatment, groups of two to three females were caged overnight with one male. Only six animals from the diabetic group became pregnant. Mating was confirmed by the presence of sperm in their morning vaginal smears and abdominal palpation around the second week of gestation. Blood glucose was measured in samples collected from the tail vein near the end of gestation. Rats which failed to get pregnant and pregnant diabetics which spontaneously recovered were excluded from the study. Pregnant rats were allowed to give birth to their litters without any interference. Offspring were immediately counted (including stillborns). Newborn weights were recorded and three pups from each dam were sacrificed by decapitation. Blood sugar was measured in samples collected from the severed neck. A laparotomy was rapidly performed and the pancreas was carefully dissected out and fixed for light and electron microscopical examination as described below. The remainder of the offspring were kept with their mother and were sacrificed at 8, 24, 72 and 168 h after delivery. Light microscopy and immunocytochemistry Pancreases were fixed in Bouin’s solution or buffered formalin, washed, dehydrated, embedded in paraffin, and sectioned (4 ,u). Formalin-fixed sections were stained TABLE I THE EFFECT OF ALLOXAN TREATMENT ON RATS FERTILITY”
Control Treated
No. of animals
Mortality
Recovered
Pregnant
% of non pregnant
6 26
0 5
0 11
6 6
0 40
a Rats treated i.v. with alloxan (40 mg/kg). Control group was treated i.v. with saline. Animals were bred three female/male. Maximum period of breeding was 4 weeks.
266
TABLE II EFFECT OF ALLOXAN TREATMENT ON MATERNAL AND FETAL GLYCEMIA”
Control Treated
Maternal glycemia (mg%)
Neonatal glycemia
before pregnancy
during pregnancy
at birth
at 8 h
114.9 i &lb 400.7 f 28.3
78.5 f 3.5 340.5 f 31.5
68.8 + 8.8 264.6 + 33.pd
111.1 + 5.4 94.9 f 4.5
a Rats were treated i.v. with alloxan (40 mgikg body weight) for 1 week before breeding. Control rats were treated with saline. Serum glucose (mg%) was measured in blood samples collected before pregnancy and in their last week of gestation. Plasma glucose level of the offspring were measured at birth and at 8 h. bValues represent the mean + SE (n = 411). ’ Significantly different from the control by independent t-test, P
263
1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/$05.00
TOXLET 02771
Endocrine pancreas in the postnatal offspring of alloxan diabetic rats
Mohamed S. Abdel-Rahman”, Fakhry A. Iskanderb “Department of Pharmacology/Toxicology,
Fatma I. Elrakhawyb and UMDNJ, New Jersey Medical School, Newark, NJ (USA)
and
bAnatomy Department, Medical School, Ain Shams University, Cairo (Egypt)
(Received 13 April 1992) (Accepted 12 June 1992) Key words: Diabetic; Postnatal; Pancreatic; Rat
SUMMARY This study was conducted to investigate the morphological changes in the postnatal pancreatic islets in offspring of diabetic rats. Diabetes was induced in female Sprague Dawley rats by intravenous injection of alloxan (40 mg/kg). After 1 week, rats with blood sugar above 270 mg% were bred and watched until spontaneous delivery occurred. Litters in the following age groups were sacrificed by decapitation, 0,8, 24, 72 and 168 h, and compared with their controls. Blood sugar levels were significantly higher in the neonates of diabetic mothers immediately after delivery compared to the control, then became normal after 8 h. Islets of the offspring of the diabetics at birth showed weak positive staining for insulin using immunocytochemical techniques. By 72 h some cells showed immunopositive staining similar to the control, while at 168 h all the p (B) cells were stained normally. /l cells of the islets from the diabetic series at birth were almost completely degranulated except for scattered granules toward the periphery. Their cytoplasm exhibited glycogen and lipid accumulations. Cells also showed signs of hyperfunction in the form of an extensive endoplasmic reticulum and well-developed Golgi complex with distended Golgi cistemae. At 8 h postnatally the population of pale secretory granules was markedly increased. The changes described at birth persisted at 24 h and, to a lesser extent, 72 h after delivery. At the age of 1 week, the /? cellsappearedto be normal.
INTRODUCTION
Pregnancy in a diabetic patient resulting in infant survival during birth was one of the great medical accomplishments of the twentieth century. Prior to the use of exogenous insulin, pregnancy in a diabetic patient was virtually unheard of, or resulted Correspondence to: M.S. Abdel-Rahman, Avenue, Newark, NJ 07103, USA.
Department of Pharmacology/Toxicology,
185 South Orange
264
in severe compromise of the patient and almost certain death of the fetus or newborn. Macrosomia, fetal distress, increased morbidity and mortality were the major complications in the outcome of diabetic pregnancies [l]. Since the discovery of the diabetogenic effect of alloxan and its specific destructive effect to the B cells of the islets, animal models have been widely used to study the effect of diabetes on pregnancy [2-51. However, decreased fertility and increased mortality of the diabetic animals complicated this approach [3,6]. Prolongation of the gestational period by 2 days was recorded [5,7]. Although macrosomia was observed in the human, conflicting reports on the fetal body weights in offspring of diabetic rats have been reported. Hultquist, Angerwall, Solomon and Lazarow et al. reported increased fetal body weight of the offspring [6,8-lo]. However, Kim et al., Lawrence and Contopoulos and Eriksson et al. reported a decrease in the rat offspring body weight [5,11,7]. In the meantime, no change in the weight of the young was reported by other investigators [12]. The insulin and somatostatin concentrations of the fetal islets of diabetic rats were sigificantly lower than the control but the glucagon level was unchanged [13,7]. A positive correlation existed between the blood sugar level of the diabetic mothers and their fetuses for a few hours after the delivery [14]. Human studies revealed that the islets of Langerhans in infants of diabetic women exhibited several morphological changes such as vacuolation of the B cells [ 151.In the meantime, an increase of the islet cell volume and marked eosinophilic infiltration of the islets were shown [16-181. The morphological changes occurring in the islets of prenatal young of diabetic rats were similar to those occurring in the adult rats subjected to hyperglycemic stress in the form of degranulation and hydropic degeneration (vacuolation of the cytoplasm) of the B cells [4]. Mild hypertrophy and hyperplasia of the islets of the newborn young of diabetic rats have also been observed [8]. Aerts and Van Assche described marked degranulation, glycogen infiltration and hyperfunction of the B cells in neonates of rats made diabetic on their first day of pregnancy [19]. These rats were treated with streptozotocin (specific B-cytotoxic drug). However, the injection of B-cytotoxic drugs has been declared to have injurious effects on the conceptus or the placenta, therefore, in an animal model an interval should elapse between the administration of the cytotoxic drug and the occurrence of pregnancy [20,7]. This study was conducted to investigate the morphological changes of the islets in offspring of alloxan diabetic rats during the first week after delivery using both light and electron microscopy. The islets cells of the young of diabetic mothers were compared with those of the neonates of control animals by using the highly specific immunocytochemical technique. In addition, attention was focused on the possible occurrence of macrosomia amongst the offspring of the diabetic rats. MATERIALS
AND METHODS
Female Sprague Dawley rats, weighing 180-255 g, were purchased from Taconic
265
Farms, Germantown, NY, and quarantined for 1 week. The rats were maintained three/cage in a temperature (25°C) and humidity (50%) controlled environment with a 12-h light/dark cycle. Laboratory rodent chow (Ralston Purina, St. Louis, MO) and water were available ad libitum. Rats were fasted for 24 h before the treatment. Twenty-six rats were injected intravenously in the tail vein with alloxan monohydrate (Sigma, St. Louis, MO), 40 mg/kg body wt. Alloxan was prepared fresh for each animal as a 5% aqueous solution. Six rats were used as controls. Animals were allowed free access to food and water immediately after treatment. Blood samples were withdrawn by cardiac puncture before treatment to determine the blood sugar using glucose oxidase [21]. Four days after treatment, non-fasting blood sugar levels were measured and rats with a blood sugar level of 260 mg% or more were used for the study. Induction and the course of pregnancy One week after treatment, groups of two to three females were caged overnight with one male. Only six animals from the diabetic group became pregnant. Mating was confirmed by the presence of sperm in their morning vaginal smears and abdominal palpation around the second week of gestation. Blood glucose was measured in samples collected from the tail vein near the end of gestation. Rats which failed to get pregnant and pregnant diabetics which spontaneously recovered were excluded from the study. Pregnant rats were allowed to give birth to their litters without any interference. Offspring were immediately counted (including stillborns). Newborn weights were recorded and three pups from each dam were sacrificed by decapitation. Blood sugar was measured in samples collected from the severed neck. A laparotomy was rapidly performed and the pancreas was carefully dissected out and fixed for light and electron microscopical examination as described below. The remainder of the offspring were kept with their mother and were sacrificed at 8, 24, 72 and 168 h after delivery. Light microscopy and immunocytochemistry Pancreases were fixed in Bouin’s solution or buffered formalin, washed, dehydrated, embedded in paraffin, and sectioned (4 ,u). Formalin-fixed sections were stained TABLE I THE EFFECT OF ALLOXAN TREATMENT ON RATS FERTILITY”
Control Treated
No. of animals
Mortality
Recovered
Pregnant
% of non pregnant
6 26
0 5
0 11
6 6
0 40
a Rats treated i.v. with alloxan (40 mg/kg). Control group was treated i.v. with saline. Animals were bred three female/male. Maximum period of breeding was 4 weeks.
266
TABLE II EFFECT OF ALLOXAN TREATMENT ON MATERNAL AND FETAL GLYCEMIA”
Control Treated
Maternal glycemia (mg%)
Neonatal glycemia
before pregnancy
during pregnancy
at birth
at 8 h
114.9 i &lb 400.7 f 28.3
78.5 f 3.5 340.5 f 31.5
68.8 + 8.8 264.6 + 33.pd
111.1 + 5.4 94.9 f 4.5
a Rats were treated i.v. with alloxan (40 mgikg body weight) for 1 week before breeding. Control rats were treated with saline. Serum glucose (mg%) was measured in blood samples collected before pregnancy and in their last week of gestation. Plasma glucose level of the offspring were measured at birth and at 8 h. bValues represent the mean + SE (n = 411). ’ Significantly different from the control by independent t-test, P
