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HISTOLOGICAL CHANGES IN THE PANCREAS, LIVERS, KIDNEYS, AND TESTES FOLLOWING THE CO-
 BACKGROUND

Diabetes mellitus is a group of metabolic diseases caused by a combination of insulin resistance and impaired insulin
secretion by pancreatic β cell. Long-lasting effect of hyperglycemia results in increased oxidative stress. Oxidative
stress results from an imbalance between radical generating and radical scavenging systems. Increased oxidative stress
has been shown to be increased in both insulin-dependent (IDDM), and noninsulin-dependent (NIDDM) diabetes
mellitus and it could cause initial β cell damage in type I diabetes, or impaired insulin production, release or function in
type II diabetes. Therefore, people with diabetes may also have greater antioxidant requirements because of increased
production of free radicals in hyperglycaemia. This present study was designed to evaluate the histological effects of
metformin and vitamin C combination therapy on pancreas, liver, kidney and testes in streptozotocin-induced diabetic
albino .

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 AIM AND OBJECTIVES OF THE STUDY

This study was aimed at evaluating the histological effects of the co-administration of metformin and
vitamin C on the pancreatic tissues, liver, kidney and testes in streptozotocin-induced diabetic male rats.

The objective of the study were to:

1. Evaluate the effect of metformin alone, vitamin C alone, and metformin and vitamin C combination
therapy in streptozotocin-induced diabetes mellitus in rats.

2. Evaluate the histological effects of metformin alone, vitamin C alone, and metformin and vitamin C
combination therapy on the liver, kidney, pancreas, and testes in streptozotocin-induced diabetic rats.

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 METHODOLOGY
• INDUCTION OF DIABETES MELLITUS

The animals were fasted overnight and diabetes was induced by a single dose intraperitoneal injection of
streptozotocin (40 mg/kg body weight) dissolved in freshly prepared 0.1M citrate buffer, pH 4.5. After
administration the animals were allowed free access to feed and water. After 48 h, the animals were tested for
diabetes using the Accu-check® Active glucometer (Roche, USA) and any animal with blood sugar level ≥ 200
mg/dl was considered diabetic (Szkudelski, 2001).
• DETERMINATION OF BLOOD GLUCOSE LEVELS

Blood samples for measuring the blood glucose level were collected from the tail tips of the fasted rats. The tail
was cleaned with methylated spirit and allowed to dry. The tail tip was pricked using a sterile lancet. One droplet
of the blood was placed on a glucose test strip and read Accu-check® active glucometer (Roche, USA). The
results obtained were recorded in mg/dl.
• HISTOPATHOLOGICAL STUDIES

Histologic study was carried out at the end of the study. The animals were sacrificed under chloroform. The
organs were carefully dissected, separated and assertion by giving them unique numbers for easy identification. 4
The organs of interest viz kidney, liver, pancreas were fixed in 10% formosaline while the testes were placed in Bouin’s
solution. After the tissues have been fixed, they were subsequently processed by passing them through different grades
of alcohol and then embedded them with paraffin wax. They were then sectioned into thin layers of three to five
microliter thick layers. This is followed by staining using haematoxylin and eosin (H & E) stain. They were
subsequently examined by the histologist using light microscope and image was captured with the digital microscope
camera. Stained tissues were viewed using an optical photomicroscope at x100 magnification.

• STATISTICAL ANALYSIS
Data were expressed as mean ± standard error of mean (S.E.M). One-way ANOVA was used to analyze data
followed by Dunnett’s post–hoc test (Graphpad prism ® 6,San Diego, USA). P<0.05 was taken as accepted level of
significant difference.

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 RESULTS

• EFFECTS OF TREATMENTS ON BLOOD GLUCOSE LEVEL

Figure 3.1 shows the mean fasting blood glucose of the treatment groups viz; metformin only, vitamin C only,
metformin and vitamin C combination, and normal control groups compared with the diabetic untreated group over the
four weeks treatment period. All the treatment groups showed a significant reduction in the blood glucose level on the
14th (p<0.01) and 28th (p<0.01) day of treatment when compared to diabetic untreated. Higher reduction in blood
glucose was recorded throughout the study in the animals treated with metformin and vitamin C combination compared
to metformin only treated group. However, the mean blood glucose level in the untreated diabetic control group was
consistently high while the blood glucose in the normal control remained within the normal glycemic range throughout
the four weeks treatment period.

• Histological changes on the pancreatic tissue, livers, kidneys and testes

Photomicrograph of the pancreas of normal rat that received 10 mg/kg of distilled water daily for 28 days (H&E,
x100) shows relatively numerous normal pancreatic islets in a background of normal exocrine glands. 6
• Photomicrograph of the pancreas of diabetic untreated rat showed a marked degeneration of the islets, with the islets

almost not visible in H & E (100X) section.

•Photomicrograph of the pancreas of diabetic rat that received 1000 mg/kg of vitamin C daily for 28 days (H&E,

100X) showed two distinct lobules of islets, visible on the histological sections amidst exocrine glands H & E, 100X.

•Photomicrograph of the pancreas of diabetic rat that received 500 mg/kg of metformin daily for 28 days. There is

scarcely any lobule of islet seen on the H & E slide (100X).

•Photomicrograph of the pancreas of diabetic rat treated with metformin 500 mg/kg and vitamin C 1000 mg/kg daily

for 28 days (H & E, 100X). Few regenerating lobules of pancreatic islet cells are seen.

• phogtomicrograph of the liver, kidney, and testes showed no significant changes after the 28th days treatment as

observed in the H and E staining section.

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 DISCUSSION
 
• In this current study, all the treatments produced significant reduction in the blood glucose levels of streptozotocin-induced
diabetic rats compared to diabetic untreated. The combination of metformin and vitamin C demonstrate a better glucose lowering
effect than metformin alone or vitamin C alone. This could be attributed to the antioxidant effect of vitamin C and the ability of
vitamin C to regenerate β-cells of the islet, and its effect on beta cell proliferation and neogenesis (Nasri et al., 2015) which was
initially destroyed by the streptozotocin. There is a continuous rise in the blood glucose levels of the untreated diabetic rats,
while the blood glucose levels of the normal control rats remained within normal glycemic level.

• Microscopic examination of the pancreatic sections of the untreated diabetic group revealed a breakdown of micro-
anatomical features including necrotic changes, β-cell degranulation, pycnotic β-cell nuclei, and severe vacuolation in
the islet. There are scarcely any visible islet cell on H and E section. This is because of the selective destruction of β-cells
by the streptozotocin (the drug used in inducing diabetes in the experimental rats). Comparison of the normal and
diabetic groups clearly shows destruction of islet cells in diabetic rats as they were irregularly shaped and atrophic.

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• In the group treated with vitamin C 1000 mg/kg alone, we observed two lobules of islets in the H & E section. This findings
suggested that some level of regeneration of the β-cells has taken place, since the experimental rats were initially treated with
Streptozotocin and consequently suffered β-cell depletion. This might be attributed to the anti-oxidant effect of vitamin C
(Shukla et al., 2012, Nasri H et al., 2015). Our findings are in agreement with the work of Lapidot et al., (2002) that the
supplements with antioxidant is used to decrease oxidative stress and improve pancreatic beta cell proliferation and function in
type 2 diabetes. Meanwhile, in the group treated with metformin 500 mg/kg alone, there was no evidence of lobules visible on
the H and E slide. This may be due to the inability of metformin to induce regeneration of islet cells. Metformin is known to exert
its anti-diabetic effect through their main mechanism of action which is to reduce hepatic glucose production by decreasing both
glyconeogenesis and glycogenolysis (wu et al., 1990).  They also increase glucose uptake by the skeletal muscle and has no
effect on β-cells regeneration.
• In the group treated with the combination of metformin 500 mg/kg and vitamin C 1000mg/kg, multiple regenerating lobules of
islets were seen on the H & E sections. This may be as a result of the ability of vitamin C to regenerate the β-cells of the islets
(Nasri et al., 2015). Comparing these two groups, metformin and vitamin C combination, and vitamin C alone, with the
untreated diabetic rats, there was evidence of recovery of β-cells of the islets, reduced fibrosepta and more obvious islet pattern
with well outlined boundaries.
• There was no comparative changes in the histopathology of the liver, kidney and testes when observed in H and E section after
the four weeks treatment period. The histology sections of these organs showed relatively normal architecture. 9
 CONCLUSION
This study was carried out to evaluate the histological effects of the co-administration of metformin and vitamin C on the
pancreatic tissues, liver, kidney and testes in streptozotocin-induced diabetic male rats.
The findings are as follows:
•The combinations of metformin (500 mg/kg) and vitamin C (1000 mg/kg) produced a more glucose lowering effect than
metformin (500 mg/kg) alone in the streptozotocin-induced diabetic rats.
•The co-administration of metformin and vitamin C possess a greater pancreatic protective effects than vitamin C alone as
evidenced by pancreatic tissue regeneration and increased islet cell mass in streptozotocin-induced diabetes in albino rats.
•There is no substantial changes in the histology of the liver, kidney and testes of the diabetic rats after 28 days treatment
period.
•This study has shown that the addition of vitamin C (an antioxidant) as an adjunct to metformin plays a crucial role in the
management of type 2 diabetes mellitus by significantly reducing blood glucose which could be related to its antioxidant
property and its ability to regenerate and restore the pancreatic Langerhans cells.

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 Lapidot, T., Walker, DM., Kanner, J. Antioxidant and pro-oxidant effects on pancreatic beta cells in Vitro. 2002;50:7220-7225.

Nasri, H., Shirzad, H., Baradaran, A., Rafieian-Kopaei, Mj. Role of antioxidant in regulating beta-cell Res Med Sci.2015 May;
20(5):419-502.

Wu, MS., Johnson, P., Sheu, WH., Hollenbeck, CB., Jeng, CY., Reaven, GM (1990). Effects of metformin on carbohydrate and
lipoproteins metabolism in NIDDM patients. Diabetes care, 13:1-8.

 Shukla, A., Priyadarshin, S., Qamar,I (2012). Involvement of vitamin c in type 2 diabetes mellitus. 2012;2:9-20.

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