Transmission Electron Microscopic (TEM) analysis of STZ-diabetes induced Cardiac Structural changes in Rats .

— Diabetes mellitus (DM) is a global health crisis, imposing a substantial burden on both healthcare systems and individuals due to its chronic nature and associated complications. Among these complications, cardiovascular diseases rank as the primary cause of morbidity and mortality in people with diabetes. Diabetic cardiomyopathy (DCM), a distinctive form of heart disease, is characterized by structural and functional abnormalities within the heart, independent of conventional cardiovascular risk factors. The multifaceted pathophysiology of DCM involves factors like hyperglycemia-induced oxidative stress, inflammation, altered myocardial metabolism, and fibrosis, collectively leading to cardiac tissue structural changes and impaired function. Streptozotocin (STZ), a compound derived from Streptomyces achromogenes, is commonly used to induce diabetes in animal models, particularly rodents, making it a valuable tool for simulating diabetes in laboratory settings. Transmission Electron Microscopy (TEM) provides a powerful means to investigate ultrastructural changes within cardiac tissues at the microscopic level, encompassing myofibrillar structure, mitochondrial integrity, sarcoplasmic reticulum, and interstitial components. Figure-1 illustrates the pathological changes caused by STZ-induced diabetes in experimental animals, showing structural alterations in cardiac and brain tissues. Notably, the use of a polyherbal extract containing Curcuma amada rhizome and Sida spinosa leaves appears to mitigate some of the detrimental effects of STZ-induced diabetes on cardiac cells, suggesting potential protective benefits.


INTRODUCTION
Diabetes mellitus (DM), a global health epidemic, poses a significant burden on both healthcare systems and individuals due to its chronic nature and associated complications.
Among these complications, cardiovascular diseases stand out as the leading cause of morbidity and mortality among individuals with diabetes.Diabetic cardiomyopathy (DCM), a distinct form of heart disease, is characterized by structural and functional abnormalities in the heart, independent of traditional cardiovascular risk factors such as hypertension and atherosclerosis.
The pathophysiology of DCM is multifaceted and includes hyperglycemia-induced oxidative stress, inflammation, altered myocardial metabolism, and fibrosis.These factors collectively contribute to structural changes within the cardiac tissue, ultimately leading to impaired cardiac function.
Streptozotocin (STZ), a naturally occurring compound derived from Streptomyces achromogenes, is widely used to induce diabetes in experimental animal models, particularly in rodents.STZ selectively damages pancreatic β-cells, resulting in insulin deficiency and hyperglycemia, making it a suitable tool for simulating
Transmission Electron Microscopy (TEM) is a powerful imaging technique that allows for the high-resolution visualization of cellular and subcellular structures.In the context of diabetic cardiomyopathy, TEM offers a valuable opportunity to explore and document ultrastructural changes within cardiac tissues at the microscopic level.This includes observing alterations in myofibrillar structure, mitochondrial integrity, sarcoplasmic reticulum, and interstitial components.
The application of TEM analysis in the study of STZinduced diabetic cardiac structural changes in rats allows for a comprehensive exploration of the ultrastructural alterations within the heart.These findings may provide critical insights into the pathogenesis of diabetic cardiomyopathy and offer valuable information for the development of targeted therapeutic interventions to mitigate cardiac complications in diabetes.

II. RESEARCH METHDOLOGY
The research methodology for conducting Transmission Electron Microscopic (TEM) analysis of STZ-diabetes induced cardiac structural changes in rats involves a systematic approach to sample preparation, imaging, and data analysis.Here is a step-by-step research methodology for your study:

Selection of Animal Model:
Choose an appropriate animal model for diabetes induction.Commonly used models include Sprague-Dawley rats.
Confirm diabetes induction using Streptozotocin (STZ) administration, and monitor blood glucose levels to confirm hyperglycemia.

Ethical Considerations:
Obtain ethical approval from the relevant institutional animal care and use committee (IACUC) or ethics board for conducting experiments on animals.
Ensure that all procedures adhere to ethical guidelines for animal welfare and humane treatment.

Sample Collection and Tissue Preparation:
Sacrifice the rats using approved euthanasia methods and collect cardiac tissue samples.
Fix the cardiac tissue samples in appropriate fixatives (e.g., glutaraldehyde) to preserve ultrastructural integrity.
Post-fix the samples with osmium tetroxide to enhance contrast for TEM imaging.

Tissue Sectioning:
Embed the fixed tissue samples in epoxy resin blocks.
Use an ultramicrotome to cut ultrathin sections (typically around 70-100 nanometers) from the embedded tissue blocks.
Transfer the ultrathin sections onto TEM grids for imaging.

TEM Imaging:
Use a transmission electron microscope equipped with an electron beam source to visualize the cardiac tissue sections at high resolution.
Capture TEM images of the cardiac tissue, focusing on areas of interest that may show structural changes, such as myofibrils, mitochondria, and cell membranes.

Data Collection and Analysis:
Analyze the TEM images to identify and document ultrastructural changes in the cardiac tissue of diabetic rats.These changes may include: • Disorganization of myofibrils.
• Changes in sarcoplasmic reticulum morphology.
• Alterations in interstitial components, including collagen and extracellular matrix.
• Quantify the observed changes and compare them with control group data, if applicable.sections from diabetic mice treated with a poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves are shown in Plate c-Group-III.These mice show a decrease in the loss and atrophy of myocytes due to STZ-diabetes, an increase in collagen fibres, and the preservation of their normal architecture.Plate d-Group-IV poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves did not produce any noticeable changes in the patients who received it.Based on the results, we may infer that the poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves, despite its apparently limited effects, may protect cardiac cells from the cytotoxicity produced by STZdiabetes.

2: Transmission electron microscopic (TEM) images of cardiac sections in control, STZ-induced diabetic, and poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves treated animals
Mice that were diabetically induced by streptozotocin (STZ) make up Group II (Plate B), whereas STZ-treated animals and those given a poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves make up Groups III and IV (Plates C and D).Initial growth (by a factor of 5,000).

Fig.3: Transmission electron microscopic images of renal sections in in control, STZ-induced diabetic, and poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves treated animals
Mice that were diabetically induced by streptozotocin (STZ) make up Group II (Plate B), whereas STZ-treated

Effects of poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves against diabetes induced lipid accumulation in and STZ-diabetic mice.
Table-3 displays the levels of total lipids (cholesterol, TGL, and phospholipids) in the hearts of the different research groups.Total cardiac lipids, cholesterol, TGL, and phospholipids were all found to be considerably (p 0.05) greater in the STZ-diabetic mice.These cardiac lipids are significantly increased or decreased in STZdiabetic mice, reflecting the abnormal lipid accumulation in the diabetic heart.Poly herbal extract of Curcuma amada rhizome and Sida spinosa leavestreated STZ-diabetic mice had a decrease in cardiac lipid accumulation.There were no discernible changes in the health of animals fed a poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves and those of normal control animals.

Table-4 displays the effects on serum and cardiac
ATPase activity of a poly herbal extract including Curcuma amada rhizome, Sida spinosa leaves, and STZ.Reduced enzyme activity in Na+/K+ ATPase and Ca2+ ATPase was seen in STZ-diabetic Group II mice compared to normal control Group I animals (p0.05).Curcuma amada rhizome and Sida spinosa leaves poly herbal extract treatment significantly improved activity levels in STZ-diabetic mice.Curcuma amada rhizome and Sida spinosa leaf poly herbal extract-treated Group IV animals did not differ significantly from normal control Group I animals.

Effect of poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves on STZ-diabetes induced urea and creatinine levels
Figure 3 shows the levels of urea and creatinine in the blood of the test animals.Diabetic mice had substantially elevated urea and creatinine levels compared to normal control animals (p 0.05).This marker was significantly reduced in Group IV mice compared to Group III diabetic mice after treatment with a poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves.The polyherbal extract of Sida spinosa leaves and Curcuma amada rhizome.The mice in Group II exhibited no significant changes on these measures.

Effects of poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves and STZ-diabetes induced lipid peroxidation
Streptozotocin (STZ) and a polyherbal extract of Curcuma amada rhizome and Sida spinosa leaves both produce lipid peroxidation in the blood and heart tissue, as illustrated graphically in Figure -4.The levels of TBARS represented the extent of lipid peroxidation.Group II-STZ diabetic mice had higher levels of TBARS in their serum and heart tissue than the control group.Compared to diabetic mice administered STZ (group-II), TBARS levels were reduced in diabetic animals given a poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves (group-III).No significant changes were seen between Group-I and Group-IV animals, who were administered a poly herbal extract consisting of Curcuma amada rhizome and Sida spinosa leaves.

IV. CONCLUSION
The study, which utilized Transmission Electron Microscopy (TEM) to analyze cardiac structural changes induced by Streptozotocin (STZ)-diabetes in rats, has provided valuable insights into the ultrastructural alterations occurring in diabetic cardiomyopathy.In conclusion, the TEM analysis has elucidated the intricate cardiac structural changes that occur in STZ-induced diabetic rats.These findings underscore the complexity of diabetic cardiomyopathy and its impact on cardiac ultrastructure.these alterations at the nanoscale level is pivotal for advancing our knowledge of this condition and identifying potential targets for therapeutic interventions.While this study contributes significantly to our understanding of diabetic cardiomyopathy, further research is needed to explore the functional consequences of these ultrastructural changes and to develop targeted treatments aimed at preserving cardiac structure and function in diabetes.
Ultimately, this research paves the way for more effective strategies to mitigate the impact of diabetic cardiomyopathy, a critical step toward improving the cardiovascular health of individuals with diabetes.
Fig.2: Transmission electron microscopic (TEM) images of cardiac sections in control, STZ-induced diabetic, and poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves treated animals

Fig. 3 :
Fig.3: Blood Urea and Creatinine levels in the control, STZ-induced diabetic, and poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves treated animals Mean standard deviation (n = 6 mice/group).a = significantly different from Group I at the 0.05 level; b = significantly different from Group II at the 0.05 level; NS = not significant.The standard for measuring urea and creatinine is milligrammes per deciliter.
deviation (n = 6 mice/group).a = significantly different from Group I at the 0.05 level; b = significantly different from Group II at the 0.05 level; NS = not significant.Superoxide dismutase (SOD) levels are reported in Units/mg protein; catalase (CAT) levels are reported in micrograms of hydrogen peroxide (H2O2) consumed (per minute) per milligramme of protein; glutathione (GSH) levels are reported in micrograms of glutathione (per minute) per milligramme of protein.

Fig. 5 :
Fig.5: Lipid peroxidation levels in serum and heart tissue in the control, STZ-induced diabetic, and poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves treated animals

of poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves on serum cardiac marker enzymes in the control and experimental group of animals.Table 1 shows
animals and those given a poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves make up Groups III and IV (Plates C and D).Initial expansion (by a factor of 5,000).Treatment with a polyherbal extract including Curcuma amada rhizome and Sida spinosa leaves restored normal kidney architecture in diabetic mice, as compared to controls.Treatment with a polyherbal extract of Curcuma amada rhizome and Sida spinosa leaves attenuated the progression of glomerulosclerosis and glomerular hypertrophy in rats exposed to STZ, and reversed the effects of STZ on glomerular function.No abnormalities or toxicity signs were seen between normal and group I control mice, which were given a poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves.Sida spinosa leaves -treated diabetic mice (group-III) had significantly lower levels of these markers.Curcuma amada rhizome and Sida spinosa leaf polyherbal extract medication control group-IV mice showed no change in LDH, CPK, CK-MB, or Troponin-I activity.

of poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves on serum lipid levels in the control and experimental group of animals.
Table-2 displays the serum cholesterol, TGL, LDL, and HDL values of control-induced and treated animals.Total lipid contents (cholesterol, TGL, and lipoproteins like LDL) increased whereas HDL levels decreased significantly (p 0.05) in STZ-diabetic mice.Changes in the levels of these lipids and lipoproteins strongly reflect abnormal lipid metabolism in STZ-diabetic mice.Poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves normalises total cholesterol, TGL, LDL, and HDL levels in STZ-diabetic mice.There were no discernible changes in the health of animals fed a poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves and those of normal control animals.

Table 2 :
Serum total cholesterol, TGL, LDL and HDL in the control, STZ-induced diabetic, and poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves treated animal NSMean standard deviation (n = 6 mice/group).a = significantly different from Group I at the 0.05 level; b = significantly different from Group II at the 0.05 level; NS = not significant.LDL, HDL, and other lipids are measured in milligrammes per millilitre of blood.

Table - 4
: Membrane bound Na + /K + -ATPase and Ca 2+ -ATPase in the normal control, diabetic control and poly herbal extract of Curcuma amada rhizome and Sida spinosa leaves treated animals NSMean standard deviation (n = 6 mice/group).a = significantly different from Group I at the 0.05 level; b = significantly different from Group II at the 0.05 level; NS = not significant.Serum Na+/K+ ATPase and Na+/K+ ATPase and Ca2+-ATPase values are reported as U/mg protein and mole phosphorus liberated/mg protein/hour, respectively.