**2. Materials and methods**

The experiments were performed on 180 adult male Wistar rats weighing from 280.0 to 300.0 g (Table 1). The animals were kept under standard vivarium conditions at pharmacology department of Volgograd Medical University, and were provided with a nutritionally balanced diet that the laboratory animals consume consistently. A pilot study was approved by the Central Regional Independent Ethics Committee (protocol № 1-06; № 43-2006; № 70- 2008; № 89-2009), and was performed in accordance with GLP when conducting preclinical studies in Russia. All animal experimentation was carried out in compliance with the International Guidelines of the European Convention for the Protection of Vertebrate Animals used in experimental studies (1997).


**Table 1.** Group distribution of experimental animals

Plasma glucose, insulin and C-peptide concentrations were measured in experimental laboratory animals. The blood glucose was determined in samples obtained from the tail vein of rats by enzymatic method, using "Glucose FKD" assay kits (Russia) on SF-46 spectrophotometer at λ=450 nm in 10 mm cuvettes. Animals with blood glucose levels >15 mmol/L were enrolled in the experiment (Akbarzadeh A. et al. 2007). Plasma insulin and Cpeptide concentrations were determined on an automated enzyme immunoassay «SUNRISE» analyzer (TECAN, Austria), using DRG Insulin ElisaKit and DRG C-peptide ElisaKit.

Alloxan-induced diabetes simulation model was developed by means of intraperitoneal administration of alloxan at a dose of 120 mg/kg. Tissue samples of animals were collected and submitted for routine histopathological investigation at 3, 7, 14 and 28 days of the experiment.

Experimental streptozotocin-induced diabetes simulation model was developed using streptozotocin (Sigma) (45 mg/kg, intravenously once a day) (Baranov V.G. 1983). Tissue samples were collected and submitted for routine histopathological investigation at 7 and 28 days of the experiment.

Experimental immune-dependent diabetes simulation model was developed by giving a subcutaneous injection of 0.2 ml of complete Freund's adjuvant (CFA) (Grand Island Biological Company, USA). Subsequently, daily intravenous injections of streptozotocin (Sigma, USA) (20 mg/kg) were given to the animals for 5 days, resulting in the development of insulindependent diabetes (Ziegler B. 1990). Tissue samples were collected and submitted for routine histopathological investigation at 3, 7, 14 and 28 days of the experiment.

Streptozotocin-nicotinamide-induced diabetes model was developed by giving an injection of streptozotocin (Sigma, USA) (intraperitoneally - 65 mg/kg citrate buffer, pH = 4.5) with a preliminary (15 minutes prior to the procedure) administration of nicotinamide (intraperitoneally - 230 mg/kg prepared in 0.9 % solution of sodium chloride) (Islam S., et al. 2009). Tissue samples were collected and submitted for routine histopathological investigation at 3, 7, 14 and 28 days of the experiment.

Pancreatic tissue was divided into three segments including intestinal, gastric and splenic parts, and then they were fixed in 10% solution of neutral buffered formalin (pH 7.4) for 24 hours. 5-6-mm thick slices were obtained on rotary microtomes and were mounted on slides. Tissue sections were stained with hematoxylin and eosin using standard histological stain techniques (Korzhevsky D.E. 2005).

For detection of α-and β- endocrine cells in the islets of Langerhans the primary antibodies against insulin and glucagon were used (Table 2). To study apoptosis, the primary antibodies to proteins, such as caspase 3, TRAIL (TNF-related apoptosis-inducing ligand), MDM2, Bcl 2, p53, Bax, NF-kB, as well as eNOS were used.


**Table 2.** IHC Primary Antibodies

226 Apoptosis and Medicine

diabetic medications.

**2. Materials and methods** 

Animals used in experimental studies (1997).

**Table 1.** Group distribution of experimental animals

ElisaKit.

experiment.

days of the experiment.

will enable us to identify specific targets for purposeful creation and development of anti-

The experiments were performed on 180 adult male Wistar rats weighing from 280.0 to 300.0 g (Table 1). The animals were kept under standard vivarium conditions at pharmacology department of Volgograd Medical University, and were provided with a nutritionally balanced diet that the laboratory animals consume consistently. A pilot study was approved by the Central Regional Independent Ethics Committee (protocol № 1-06; № 43-2006; № 70- 2008; № 89-2009), and was performed in accordance with GLP when conducting preclinical studies in Russia. All animal experimentation was carried out in compliance with the International Guidelines of the European Convention for the Protection of Vertebrate

Intact control 3, 7, 14, 28 40 Alloxan-induced diabetes 3, 7, 14, 28 40 Streptozotocin-induced diabetes 7, 28 20 Immune-dependent diabetes 3, 7, 14, 28 40 Streptozotocin-nicotinamide-induced diabetes 3, 7, 14, 28 40

Plasma glucose, insulin and C-peptide concentrations were measured in experimental laboratory animals. The blood glucose was determined in samples obtained from the tail vein of rats by enzymatic method, using "Glucose FKD" assay kits (Russia) on SF-46 spectrophotometer at λ=450 nm in 10 mm cuvettes. Animals with blood glucose levels >15 mmol/L were enrolled in the experiment (Akbarzadeh A. et al. 2007). Plasma insulin and Cpeptide concentrations were determined on an automated enzyme immunoassay «SUNRISE» analyzer (TECAN, Austria), using DRG Insulin ElisaKit and DRG C-peptide

Alloxan-induced diabetes simulation model was developed by means of intraperitoneal administration of alloxan at a dose of 120 mg/kg. Tissue samples of animals were collected and submitted for routine histopathological investigation at 3, 7, 14 and 28 days of the

Experimental streptozotocin-induced diabetes simulation model was developed using streptozotocin (Sigma) (45 mg/kg, intravenously once a day) (Baranov V.G. 1983). Tissue samples were collected and submitted for routine histopathological investigation at 7 and 28

**Group Day of experiment Amount of** 

**animals** 

Immunohistochemistry was performed according to manufacturers' protocols using ABC (Novocastra, UK), «UltraVision» (Lab Vision, UK) and «EnVision» (Dako Cytomation, Denmark) antibody detection systems and a chromogen, diaminobenzidine under the protocol on high temperature antigen unmasking technique using «*Pascal*» mini autoclave (Dako Cytomation, Denmark) (Kumar G.L. et al. 2009). The reliability of the obtained results was defined using both positive and negative control antigens, as well as negative control antibodies.

Pancreatic Islet Beta-Cell Apoptosis in Experimental Diabetes Mellitus 229

**Intestinal Gastric Splenic** 

3 6,8±2,5 12,6±6,0 13,8±7,3 7 6,7±2,2 12,0±4,5 12,9±2,1 14 6,9±3,1 12,1±5,2 14,0±2,9 28 6,6±4,2 12,8±4,5 14,3±5,2

3 51,3±9,2 64,6±9,1 **78,2±6,1\*\***  7 53,8±7,6 63,4±7,0 **75,4±8,7\*\***  14 52,1±5,5 64,5±8,7 **79,4±4,6\*\***  28 54,3±5,4 66,2±3,6 **77,3±4,3\*\*** 

3 37,4±5,2 31,2±3,5 **22,2±3,4\*\***  7 39,3±4,3 30,4±4,0 **21,3±3,0\*\***  14 38,5±3,5 32,4±3,2 **19,7±2,3\*\***  28 37,1±1,0 30,2±1,2 **17,9±1,3\*\*** 

segments of the pancreas (Fig. 2A). The maximum α-endocrine cell area was determined in the intestinal and gastric segments of the pancreas; however, this area was statistically significant lower (p <0.05) in the splenic segment (Table 3). There was a statistically significant (p ≤ 0.05) direct relationship between the total volume fraction of pancreatic islets and the area of βendocrine cells depending on their location. The amount of islets and β-endocrine cells tended

**Figure 1.** Blood glucose dynamics (mmol/L) in rats with different models of experimental diabetes. \* -

**Indicator Day of experiment Segment of the pancreas** 

Significant changes compared with the intact control group.

Volume fraction of islets,%

Total area of β-

Total area of α-

cells,%

cells,%

to increase in the following segments of the pancreas: intestinal →gastric → splenic.

Immunohistochemical reaction was based on visual evaluation, taking into account the intensity of color, or on determination of the specific amount of positively stained cells (Allred DC, et al. 1998).

Photo images were captured with an «AxioScope» microscope (Carl Zeiss, Germany) and a «PowerShot» digital camera (Canon, Japan). Morphometric analysis was performed using "VideoTestMorfo-4" software (Russia). In the course of the study we determined the relationship between the total α-and β- endocrine cell area and the total area of the islet (S,%), between the volume fraction (ML, %) of islets and exocrine glands, as well as the area of β-cell nuclei (S, µm2). We also measured apoptotic index (AI), i.e. the relative amount of β-endocrine cells with apoptotic structural and immunohistochemical changes.

The research results were processed using basic statistical analysis techniques as well as "Video TestMorfo-4», Excel Microsoft Office (Microsoft, USA) and STATISTICA 6.0 software (StatSoft Inc., USA). The analysis of the parameters for normally-distributed values was performed using Student's t test. Nonparametric statistics was calculated using Mann-Whitney test. To compare qualitative variables, the chi-square test and the Fisher exact test were used. Differences were considered significant if error probability was p <0.05.
