Experimental Analysis of Tear Fluid and Its Processing for the Diagnosis of Multiple Sclerosis.

A pilot analysis of the tear fluid of patients with multiple sclerosis (MS) collected by glass microcapillary was performed using various experimental methods: liquid chromatography-mass spectrometry, Raman spectroscopy, infrared spectroscopy, and atomic-force microscopy. Infrared spectroscopy found no significant difference between the tear fluid of MS patients and the control spectra; all three significant peaks were located at around the same positions. Raman analysis showed differences between the spectra of the tear fluid of MS patients and the spectra of healthy subjects, which indicated a decrease in tryptophan and phenylalanine content and changes in the relative contributions of the secondary structures of the polypeptide chains of tear proteins. Atomic-force microscopy exhibited a surface fern-shaped dendrite morphology of the tear fluid of patients with MS, with less roughness on both oriented silicon (100) and glass substrates compared to the tear fluid of control subjects. The results of liquid chromatography-mass spectrometry showed downregulation of glycosphingolipid metabolism, sphingolipid metabolism, and lipid metabolism. Proteomic analysis identified upregulated proteins in the tear fluid of patients with MS such as cystatine, phospholipid transfer protein, transcobalamin-1, immunoglobulin lambda variable 1-47, lactoperoxidase, and ferroptosis suppressor protein 1; and downregulated proteins such as haptoglobin, prosaposin, cytoskeletal keratin type I pre-mRNA-processing factor 17, neutrophil gelatinase-associated lipocalin, and phospholipase A2. This study showed that the tear proteome in patients with MS is modified and can reflect inflammation. Tear fluid is not a commonly used biological material in clinico-biochemical laboratories. Experimental proteomics has the potential to become a promising contemporary tool for personalized medicine, and it might be applied in clinical practice by providing a detailed analysis of the tear-fluid proteomic profile of patients with MS.

Flavonoids as Promising Natural Compounds in the Prevention and Treatment of Selected Skin Diseases.

Phytochemicals represent a large and diverse group of naturally occurring compounds, bioactive nutrients, or phytonutrients produced by plants, widely found in fruits, vegetables, whole grains products, legumes, beans, herbs, seeds, nuts, tea, and dark chocolate. They are classified according to their chemical structures and functional properties. Flavonoids belong to the phenolic class of phytochemicals with potential solid pharmacological effects as modulators of multiple signal transduction pathways. Their beneficial effect on the human body is associated with their antioxidant, anti-inflammatory, antimutagenic, and anticarcinogenic properties. Flavonoids are also widely used in various nutritional, pharmaceutical, medical, and cosmetic applications. In our review, we discuss the positive effect of flavonoids on chronic skin diseases such as vitiligo, psoriasis, acne, and atopic dermatitis.

The Effects of Fisetin on Cyclosporine-Treated Dry Eye Disease in Dogs.

Dry eye disease (DED) is a chronic debilitating ophthalmological disease with the current therapeutic options focused on the suppression of the symptoms. Among the possibilities of how to improve DED therapy, polyphenols have shown an enormous capacity to counteract DED functional changes. The study aimed to specifically target pathophysiological mechanisms by the addition of fisetin to the cyclosporine treatment protocol. We examined dog patients with DED on cyclosporine treatment that were administered 0.1% fisetin or fisetin-free eye drops. For the assessment of fisetin effects, tear film production and matrix metalloproteinase 9 (MMP-9) were studied in the tear film. Tear production was not recovered after 7 or 14 days (9.40 mm ± 6.02 mm, p = 0.47; 9.80 mm ± 6.83 mm, p = 0.53, respectively). MMP-9 levels significantly increased after 7 days and then dropped after 14 days (775.44 ng/mL ± 527.52 ng/mL, p = 0.05; 328.49 ng/mL ± 376.29 ng/mL, p = 1.00, respectively). Fisetin addition to cyclosporine DED treatment was not able to restore tear fluid production but influenced molecular pathological events through MMP-9.

Derivative Three-Dimensional Synchronous Fluorescence Analysis of Tear Fluid and Their Processing for the Diagnosis of Glaucoma.

BACKGROUND: Sensitive and rapid diagnosis of the early stages of glaucoma from tear fluid is a great challenge for researchers. METHODS: Tear fluid was analyzed using three-dimensional synchronous fluorescence spectroscopy (3D-SFS). Our previously published results briefly describe the main methods which applied the second derivative to a selected synchronous spectrum Δλ = 110 nm in distinguishing between healthy subjects (CTRL) and patients with glaucoma (POAG). RESULTS: In this paper, a novel strategy was used to evaluate three-dimensional spectra from the tear fluid database of our patients. A series of synchronous excitation spectra were processed as a front view and presented as a single curve showcasing the overall fluorescence profile of the tear fluid. The second derivative spectrum provides two parameters that can enhance the distinction between CTRL and POAG tear fluid. CONCLUSIONS: Combining different types of 3D-SFS data can offer interesting and useful diagnostic tools and it can be used as input for machine learning and process automation.

Salivary Redox Homeostasis in Human Health and Disease.

Homeostasis is a self-regulatory dynamic process that maintains a stable internal environment in the human body. These regulations are essential for the optimal functioning of enzymes necessary for human health. Homeostasis elucidates disrupted mechanisms leading to the development of various pathological conditions caused by oxidative stress. In our work, we discuss redox homeostasis and salivary antioxidant activity during healthy periods and in periods of disease: dental carries, oral cavity cancer, periodontal diseases, cardiovascular diseases, diabetes mellitus, systemic sclerosis, and pancreatitis. The composition of saliva reflects dynamic changes in the organism, which makes it an excellent tool for determining clinically valuable biomarkers. The oral cavity and saliva may form the first line of defense against oxidative stress. Analysis of salivary antioxidants may be helpful as a diagnostic, prognostic, and therapeutic marker of not only oral, but also systemic health.

Tackling endothelium remodeling in cardiovascular disease.

Endothelial dysfunction is considered an early marker of atherosclerosis. Herein, we address the molecular mechanisms affecting endothelium remodeling in disease. Vascular calcification is highly prevalent in patients with ischemic cardiovascular disease, cerebrovascular disorder, and renal failure, being a common feature in aging, diabetes, dyslipidemia, abnormal valve biomechanics, end-stage renal disease and atherosclerosis, a major cause of mortality and morbidity. Oxidative stress promotes calcification of vascular smooth muscle cells (SMC) by increasing osteogenic transcription factors expression and activity in atherosclerotic plaques. Various markers of osteogenic differentiation are expressed by SMC in calcified atherosclerotic lesions. Interestingly, decreased levels of some bone factors and microRNAs accelerate vascular calcification and injured tissue regeneration. Another key player in endothelial remodeling is amino acids metabolism. Branched-chain amino acids are catabolized in several nonhepatic tissues including cardiac muscle. Immune activation and inflammation in cardiovascular disease patients associate with higher phenylalanine/tyrosine ratios. Understanding the whole process that underlies endothelium dysfunction is of paramount importance for the development of new therapeutic approaches.

Stage-Dependent Levels of Brain-Derived Neurotrophic Factor and Matrix Metalloproteinase 9 in the Prognosis of Colorectal Cancer.

PURPOSE: The development of sensitive and non-invasive biomarkers for the early detection of CRC and determination of their role in the individual stages of CRC. METHODS: MMP-9 expression in serum and tissue, and BDNF expression in plasma were detected using the ELISA method. MMP-9 and BDNF in the tissue were also determined by immunohistochemical staining. RESULTS: To assess the balance between changes in survival and tumor progression, we compared BDNF/MMP-9 ratios in tissues of living and deceased individuals. The tissue BDNF/MMP-9 ratio (evaluated immunohistochemically) decreased significantly with the progression of the disease in living patients. The BDNF/MMP-9 ratio was statistically significantly reduced in stages II and III compared to the benign group. However, in deceased individuals, the ratio showed an opposite tendency. CONCLUSION: The determination of the tissue BDNF/MMP9 ratio can be used as a prognostic biomarker of CRC.

Effect of tear fluid sampling and processing on total protein quantity and electrophoretic pattern.

Human tears contain more than 1500 proteins that could be diagnostically relevant. To date, numerous candidates on a biomarker of protein origin were identified for ocular and systemic diseases. However, the suitable sampling method is still the subject of discussion. To address the need for a description of sampling methods properties for possible clinical analyses, we studied a total protein concentration and electrophoretic pattern of tear fluid collected by capillary tubes, Schirmer strips, cellulose microsponges, and flushing. The total protein concentration was 4.339 µg/µL ± 1.905 µg/µL, 0.967 µg/µL ± 0.117 µg/µL, 0.022 µg/µL ± 0.016 µg/µL, and 0.008 µg/µL ± 0.006 µg/µ for the capillary tubes, Schirmer strips, flushing, and cellulose microsponges, respectively. Sodium dodecyl sulfate polyacrylamide electrophoresis showed the different patterns of tear proteins obtained by the above-mentioned sampling methods. These differences could originate from the use of a bigger amount of extraction reagent that was not used in the case of capillary tubes, and retention of the proteins by strips and sponges. Taken together, capillary tubes, Schirmer strips, cellulose microsponges, and flushing represent sensitive and convenient sampling methods for tear fluid collection. For the isolation of proteins from strips and sponges, and for the flushing, less than 100 µL of a reagent should be used to ensure the sufficient concentration of the biomarkers in a trace amount.

Native fluorescence of tear fluid as a tool for diagnostics of glaucoma.

Glaucoma is one of the leading causes of irreversible vision loss worldwide. There is an enormous need for the detection of its early stages and also speeding up and simplifying regular examinations. Among the new diagnostic approaches, the use of tear fluid has been intensively investigated in recent years. For this purpose, we analyzed the tear fluid of patients with glaucoma and related diseases. To sensitively capture the subtle ocular abnormalities related to glaucoma and manifested in tear fluid, we used synchronous fluorescence spectroscopy. In this observational case-control study, we detected significant differences in the intensity of tear fluid fluorescence located at λ ex/Δλ = 280/70 nm between the groups of primary open-angle glaucoma (p < 0.01), suspected glaucoma (p < 0.0001), and ocular hypertension (p < 0.05), when compared to the healthy control group. The signal was not significantly higher in women than in men (p = 0.05), and no correlation was found with age (r = -0.05, p > 0.05), nor treatment (p > 0.05). Taken together, tear fluid fluorescence could serve as a discriminative parameter between patients with glaucoma, related diseases, and healthy control subjects and might contribute to the improvement of diagnostics of these diseases.

Evidence of Polyphenols Efficacy against Dry Eye Disease.

Dry eye disease is a multifactorial pathology compromising the quality of life of patients, resulting in significant damage of the ocular surface and discomfort. The current therapeutical strategies are not able to definitively resolve the underlying causes and stop the symptoms. Polyphenols are promising natural molecules that are receiving increasing attention for their activity/effects in counteracting the main pathologic mechanisms of dry eye disease and reducing its symptoms. In the present review, a deep literature search focusing on the main polyphenols tested against dry eye disease was conducted, analyzing related in vitro, in vivo, and clinical studies to provide a comprehensive and current review on the state of the art. Polyphenols present multiple effects against dry eye diseases-related ocular surface injury. In particular, the observed beneficial effects of polyphenols on corneal cells are the reduction of the pathological processes of inflammation, oxidative stress, and apoptosis and modulation of the tear film. Due to numerous studies reporting that polyphenols are effective and safe for treating the pathological mechanisms of this ocular surface disease, we believe that future studies should confirm and extend the evidence of polyphenols efficacy in clinical practice against dry eye disease and help to develop new ophthalmic drug(s).

Tear fluid biomarkers in major depressive disorder: Potential of spectral methods in biomarker discovery.

Spectroscopic methods represent a group of analytical methods that demonstrate high potential in providing clinically relevant diagnostic information, such as biochemical, functional or structural changes of macromolecular complexes that might occur due to pathological processes or therapeutic intervention. Although application of these methods in the field of psychiatric research is still relatively recent, the preliminary results show that they have the capacity to detect subtle neurobiological abnormalities in major depressive disorder (MDD). Methods of mass spectrometry (MALDI-TOF MS), zymography, synchronous fluorescence spectroscopy (SFS), circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM) were used to analyze the human tear fluid of subjects with MDD. Using MALDI-TOF MS, two diagnostically significant peaks (3747 and 16 411 m/z) were identified with an AUC value of 0.89 and 0.92 in tear fluid of subjects with MDD vs controls, respectively. We also identified various forms of matrix metalloproteinase 9 in subjects with MDD using zymography and synchronous fluorescence spectra (SFS) showed a significant increase in fluorescence intensity at 280 nm. CD spectra were redshifted in tear fluid of subjects with MDD vs healthy controls. FTIR spectroscopy showed changes in the positions of peaks for amide A, I, II in tear fluid of subjects with MDD vs controls. Moreover, atomic force microscopy (AFM) showed different pattern in the crystal structures of tear fluid components in subjects with MDD. SFS, CD, FTIR spectroscopy, AFM and MALDI-TOF MS confirmed, that the human tear fluid proteome could be helpful in discriminating between the group of subjects with MDD and healthy controls. These preliminary findings suggest that spectral methods could represent a useful tool in clinical psychiatry, especially in establishing differential diagnosis, monitoring illness progression and the effect of psychiatric treatment.

Low-level laser therapy for protection against skeletal muscle damage after ischemia-reperfusion injury in rat hindlimbs.

BACKGROUND AND OBJECTIVE: Despite numerous hypotheses regarding the action of laser light, the use of low-level laser therapy (LLLT) in ischemic reperfusion (I/R) injury is still being verified. The present study investigates the effects of low-level laser irradiation (LLLI) on I/R injury of the musculus gracilis in rats. MATERIALS AND METHODS: I/R injury of the musculus gracilis flap was induced in male adult Sprague-Dawley rats (n = 84). Rats were subdivided depending on treatment into four subgroups: (1) healthy group, (2) I/R injury without irradiation, (3) R group irradiated only during reperfusion after injury, and (4) IR group irradiated during ischemia and reperfusion injury. LLLT (AlGaInP; λ = 670 nm; 4 J/cm²; 40 mW/cm²) was applied to the injured muscle four times daily until euthanasia. RESULTS: Lactate dehydrogenase (LD) levels were significantly lower (P<0.05) in the irradiated groups during the first 12-120 hours, while the lower creatine kinase (CK) level reached statistical significance only at 24 hours in the irradiated group when compared to the control group. The number of polymorphonuclear leukocytes in the gracilis muscle was significantly lower in the treated group only on the second day (P<0.0001). The lowered percentage of necrosis in the muscle tissue was statistically significant after 6 and 10 days of treatment (P<0.0001), while lower atrophy and higher neovascularization were observed at 6-14 days of irradiation (P<0.05). There was no statistically significant difference between the group irradiated only during reperfusion and that irradiated during ischemia and reperfusion. CONCLUSION: LLLT confers a protective effect against early inflammatory tissue response, further atrophy, and necrosis of the muscle and it stimulates neovascularization after I/R injury.

Nuclear factor-kB and nitric oxide synthases in red blood cells: good or bad in obesity? A preliminary study.

Emerging evidence suggests that red blood cells (RBCs) are involved in many functions essential for life. Nuclear factor-kB (NF-kB), nitric oxide synthases (inducible nitric oxide synthase -iNOS-, endothelial nitric oxide synthase -eNOS-) and interleukin-1ß (-IL-1ß-) are all proteins that have been identified in RBCs. In nucleated cells, such as white blood cells (WBCs), these proteins have well investigated roles, linked to stress and inflammation. It is not the same in erythrocytes, for this reason, we considered obese patients for studying the morphology of RBCs. We studied a possible correlation between their morphological changes and several protein expressions. Moreover, we compared the results about the aforementioned proteins and antioxidant markers with those obtained in WBCs from healthy and obese patients before and after omega-3 polyunsaturated fatty acid supplementation. This latter scientific point is important in order to determine whether there are differences in the expression of nucleated and anucleated cells. The morphology of RBCs changed in obese patients, but it is significantly restored after six weeks of supplementation. The expression of antioxidant enzymes changed in RBCs and WBCs in obesity but all proteins restore their positivity after supplementation. We found that: the presence of NF-kB, antioxidant enzymes and eNOS in healthy RBCs could indicate a role of these proteins as regulators of cellular metabolism; obese WBCs showed a higher NF-kB, iNOS and IL-1ß positivity, whereas eNOS presence did not significantly change in these cells. We tried to explain the different positivity of NF-kB, proposing a dual role for this protein, as prolifespan and as proinflammatory processes, depending on examined cells. In conclusion, we have considered the literature that focuses on the omega-6/omega-3 ratio. The ratio changed from the past, especially in people whose diet is strongly westernized worsening the state of health of the patient and leading to an higher incidence of obesity. Our study hypothesizes that the supplementation could help to restore the correct ratio.

Noninvasive diagnostic methods for diabetes mellitus from tear fluid.

Diabetes mellitus and prolonged hyperglycemia can cause diabetic retinopathy. Diabetic retinopathy arises from damage to retinal vessels and, in its final stages, causes blindness. The early stages are often asymptomatic and although regular screening of diabetic patients is recommended, the beginning of diabetic retinopathy is insufficiently detected. The diagnostic potential of fluorescence spectroscopy, infrared spectroscopy and atomic force microscopy as the untraditional methods for diabetes mellitus was investigated using tear fluid. In our pilot study the structural changes of tear fluid of patients with diabetes mellitus after insulin and oral antidiabetic drug treatment was compared with healthy subjects. The results of analysis, infrared spectroscopy and atomic force microscopy confirmed structural changes in tear fluid of patients in comparison with the tear fluid of healthy subjects. Using new experimental laboratory methods in future could contribute to an improvement in diagnosis of diabetes mellitus and other selected ocular diseases using tear fluid.

Diagnostic potential of tears in ophthalmology.

In research circles, there is an increasing need to seek and identify new methods in the diagnosis of pathologies, monitoring the progression of the disease and response to treatment. The sensitivity of detection technologies has improved markedly, and enables the quantification of analyses in very small quantities. Tears represent a biological material with ever increasingly developing possibilities in the diagnosis of various pathologies. Our objective was to compile a basic overview of the diagnostic potential of tears via a summary of the potential lachrymal biomarkers of various pathologies. The article contains descriptions of protein biomarkers studied particularly in recent years, which correlate with a certain ocular pathology (dry eye, allergy, glaucoma etc.). It also summarises the results published to date in the field of systemic pathologies in patients with scleroderma, cystic fibrosis, diabetes mellitus, multiple sclerosis, cancers and Parkinson's disease. It concentrates on proteomic analyses, with the aim of improving the effectiveness of markers which could be used in future also in the timely diagnosis of ocular pathologies in clinical practice.Key words: Tears, proteins, biomarker, ocular pathologies, systemic pathologies.

Comparison of the effects of selected chalcones, dihydrochalcones and some cyclic flavonoids on mitochondrial outer membrane determined by fluorescence spectroscopy.

The effect on mitochondrial outer membrane of 4-hydroxychalcone (1), the cyclic chalcone analogues E-2-(4'-hydroxybenzylidene)-1-indanone (2a) and E-2-(4'-hydroxybenzylidene)-1-tetralone (2b), the dihydrochalcones phloretin (3a) and phloridzin (3b), the flavanones naringenin (4a) and naringin (4b), and the flavonol quercetin (5) was investigated by fluorescence spectroscopy. Excitation and emission fluorescence spectra of each flavonoid and synthetic analogue were recorded in respiration medium containing 1 mM succinate. Initial interaction of the compounds with the outer mitochondrial membrane was investigated by recording their fluorescence polarization in the presence of rat liver mitochondria. Most of the compounds displayed an elevated fluorescence polarization on mixing with mitochondria at the zero time point. During the investigated 20 min period the initial fluorescence polarization values remained constant (1, 2a), or a gradual depression of the measured polarization values could be observed (2b, 3a, 4b, 5). In the case of naringenin (4a), however, similar to the previously investigated seven-membered cyclic chalcone analogue E-2-(4 -methoxybenzylidene)-1-benzosuberone, a slight, continuous increase of fluorescence polarization could be detected during the 20 min experiment. Phloridzin (3b) showed an increased fluorescence polarization in first 10 min, which was slightly depressed by the 20 min time point.

Antiproliferative effect of ß-escin - an in vitro study.

This study examined the antiproliferative effects of ß-escin (E) in cancer cells. The study showed that E inhibited cancer cells growth in a dose-dependent manner. The flow cytometric analysis revealed an escin-induced increase in the sub-G1 DNA content, which is considered to be a marker of apoptosis. Apoptosis was also confirmed by annexin V staining and DNA fragmentation assay. These effects were associated with increased generation of reactive oxygen species (ROS), caspase-3 activation and decreased mitochondrial membrane potential (MMP). Moreover, escin decreased mitochondrial protein content and mitochondrial fluorescence intensity as well as caused depletion of glutathione (GSH). However, activity of glutathione peroxidase (GPx) and glutathione reductase (GR) was not significantly changed in escin-treated cells. In conclusion, our results demonstrated that E has apoptotic effects in human cancer cells through the mechanisms involving mitochondrial perturbation. Although the exact mechanism needs to be investigated further, it can be concluded that E may be a useful candidate agent for cancer treatment.

Comparison of effect of selected synthetic chalcone analogues on mitochondrial outer membrane determined by fluorescence spectroscopy.

Effect on mitochondrial outer membrane of six selected synthetic cyclic chalcone analogues, E-2-arylmethylene-1-tetralones (2) and E-2-arylmethylene-1-benzosuberones (3), were investigated by fluorescence spectroscopy. The selected compounds represent derivatives with different degree of cytotoxicity against murine and human cancer lines. Excitation and emission fluorescence spectra of the cyclic chalcone analogues 2 and 3 were recorded in respiration medium containing 1 mM succinate. It was found that the ring size as well as the nature and location of the aromatic substituents have significant effect on fluorescence of the compounds. Interaction of subtoxic concentration of compounds 2 and 3 with the outer mitochondrial membrane was investigated by recording their fluorescence polarization in the presence of rat liver mitochondria. The most cytotoxic E-2-(4'-methoxybenzylidene)-1-benzosuberone (3b) was found to display a continuous increase of fluorescence polarization signal in the presence of mitochondria--a different pattern of interaction with the mitochondrial outer membrane from that observed for rest of the investigated compounds.

Trehalase as a possible marker of intestinal ischemia--reperfusion injury.

BACKGROUND: Different pathological affections of the small intestine cause corresponding morphological and functional changes. The present study was aimed to assess intestinal trehalase activities during ischemia and following reperfusion, correlate them with the pathological changes and determine whether trehalase could be used as a biochemical marker of the intestinal ischemia, ischemia - reperfusion injury. MATERIAL AND METHODS: Wistar rats, randomly divided into 5 experimental groups (IR) (each n=15), were subjected to one hour mesenteric ischemia followed by 0, 1, 4, 12 and 24 hours of reperfusion. As a control group sham operated animals were used (n=15). The activity of trehalase was determined using an adapted Dahlqwist method. The range of intestinal injury was determined using histological (histopathological injury index and goblet cell quantification) and immunohistochemical (Ki67, InSitu TUNEL) methods. RESULTS: The highest activities of trehalase were recorded in the control group (C=4.42 ± 0.373 µmol/mg/h). The most altered intestinal histology detected in group IR1 was accompanied by the lowest trehalase activity (IR1=0.97 ± 0.209 µmol/mg/h; p < 0.001 C vs. IR1). Improved histological structure in the remaining reperfusion periods correlated with increase in trehalase activity. Almost normal mucosal histological architecture and 72% of the enzymatic activity were restored after 24 hours of reperfusion (IR24=3.20 ± 0.266 µmol/mg/h; p < 0.01 IR1 vs. IR24). CONCLUSION: The correlation between intestinal histology and trehalase activities during intestinal injury has been shown. Trehalase activity is closely associated with the status of the histological architecture of the small intestine.

Intravenous administration of tetramethylpyrazine reduces intestinal ischemia-reperfusion injury in rats.

Intestinal ischemia-reperfusion injury (IIRI) is a life-threatening condition requiring prompt medical intervention. Tetramethylpyrazine (TMP) is a biologically active alkaloid isolated from Ligusticum wallichii. Previously, it was shown that TMP causes vasodilatation and inhibition of platelet aggregation as well as exhibits significant antioxidant effects. Therefore, the aim of the present study was to evaluate possible therapeutic effects of TMP in the prevention of IIRI. Wistar rats (n = 80) were randomly divided into eight experimental groups and subjected to a 1 h occlusion of cranial mesenteric artery followed by 0, 1, 12, and 24 h period of reperfusion. Thirty minutes before the IIRI animals received either TMP (30 mg/kg, i.v.) or identical volume of saline. In addition, a control group of 10 animals was not exposed to IIRI. Intestine morphology was evaluated by using histopathological injury index examination (HII), goblet and Paneth cells quantification as well as by applying immunofluorescent methods such as InSitu TUNEL and caspase-3 positivity assessment. Here we showed that preconditioning with TMP prior IIRI decreases the grade of injury. Significant reduction of HII was detected in TMP pretreated groups after 0, 1, and 12 h of reperfusion where injury reduction up to 75% was found. Lower histopathological damage in preconditioned groups was accompanied with increased number of secretory epithelial cells and decreased number of apoptotic cells. These results demonstrate the protective effect of TMP on the small intestine mucosa, suggesting administration of TMP as a molecule for pharmacological intervention against IIRI.