Association between gut health and gut microbiota in a polluted environment.

Animals host complex bacterial communities in their gastrointestinal tracts, with which they share a mutualistic interaction. The numerous effects these interactions grant to the host include regulation of the immune system, defense against pathogen invasion, digestion of otherwise undigestible foodstuffs, and impacts on host behaviour. Exposure to stressors, such as environmental pollution, parasites, and/or predators, can alter the composition of the gut microbiome, potentially affecting host-microbiome interactions that can be manifest in the host as, for example, metabolic dysfunction or inflammation. However, whether a change in gut microbiota in wild animals associates with a change in host condition is seldom examined. Thus, we quantified whether wild bank voles inhabiting a polluted environment, areas where there are environmental radionuclides, exhibited a change in gut microbiota (using 16S amplicon sequencing) and concomitant change in host health using a combined approach of transcriptomics, histological staining analyses of colon tissue, and quantification of short-chain fatty acids in faeces and blood. Concomitant with a change in gut microbiota in animals inhabiting contaminated areas, we found evidence of poor gut health in the host, such as hypotrophy of goblet cells and likely weakened mucus layer and related changes in Clca1 and Agr2 gene expression, but no visible inflammation in colon tissue. Through this case study we show that inhabiting a polluted environment can have wide reaching effects on the gut health of affected animals, and that gut health and other host health parameters should be examined together with gut microbiota in ecotoxicological studies.

The effect of C60 fullerene on the mechanokinetics of muscle gastrocnemius contraction in chronically alcoholized rats.

The C60 fullerene effect (oral administration at a dose of 1 mg kg-1) on the selected biomechanical parameters of muscle gastrocnemius contraction, biochemical indicators of blood and muscle tissue as well as histological changes in rat muscle tissue after chronic alcoholization for 3, 6 and 9 months was studied in detail. Water-soluble C60 fullerenes were shown to reduce the pathological processes development in the muscle apparatus by an average of (35-40)%. In particular, they reduced the time occurrence of fatigue processes in muscle during the long-term development of alcoholic myopathy and inhibited oxidative processes in muscle, thereby preventing its degradation. These findings open up the possibility of using C60 fullerenes as potent antioxidants for the correction of the pathological conditions of the muscle system arising from alcohol intoxication.

The long-term consequences of antibiotic therapy: Role of colonic short-chain fatty acids (SCFA) system and intestinal barrier integrity.

Epidemiological studies revealed that antibiotics exposure increases a risk of inflammatory bowel diseases (IBD) development. It remained largely unknown how antibiotic-induced dysbiosis confers the risk for enhanced inflammatory response. The aim of the present study was to test the hypothesis that SCFAs, their receptors and transporters mediate the antibiotic long-term effects on the functional state of colonic mucosa and susceptibility to the experimental colitis. Male Wistar rats were treated daily for 14 days with antibiotic ceftriaxone (300 mg/kg, i.m.) or vehicle; euthanized by CO2 inhalation followed by cervical dislocation in 1, 14 or 56 days after antibiotic withdrawal. We found increased cecum weight and sustained changes in microbiota composition after ceftriaxone treatment with increased number of conditionally pathogenic enterobacteria, E. coli, Clostridium, Staphylococcus spp. and hemolytic bacteria even at 56 days after antibiotic withdrawal. The concentration of SCFAs was decreased after ceftriaxone withdrawal. We found decreased immunoreactivity of the FFA2, FFA3 receptors, SMCT1 and increased MCT1 & MCT4 transporters of SCFAs in colon mucosa. These changes evoked a significant shift in colonic mucosal homeostasis: the disturbance of oxidant-antioxidant balance; activation of redox-sensitive transcription factor HIF1α and ERK1/2 MAP kinase; increased colonic epithelial permeability and bacterial translocation to blood; morphological remodeling of the colonic tissue. Ceftriaxone pretreatment significantly reinforced inflammation during experimental colitis 56 days after ceftriaxone withdrawal, which was confirmed by increased histopathology of colitis, Goblet cell dysfunction, colonic dilatation and wall thickening, and increased serum levels of inflammatory cytokines (TNF-α and IL-10). Since the recognition of the importance of microbiota metabolic activity rather than their composition in the development of inflammatory disorders, e.g. IBD, the present study is the first report on the role of the SCFA system in the long lasting side effects of antibiotic treatment and its implication in IBD development.

Na+/H+ exchanger 1 inhibition reverses manifestation of peripheral diabetic neuropathy in type 1 diabetic rats.

Evidence for an important role for Na(+)/H(+) exchangers in diabetic complications is emerging. The aim of this study was to evaluate whether Na(+)/H(+) exchanger 1 inhibition reverses experimental peripheral diabetic neuropathy. Control and streptozotocin-diabetic rats were treated with the specific Na(+)/H(+) exchanger 1 inhibitor cariporide for 4 wk after 12 wk without treatment. Neuropathy end points included sciatic motor and sensory nerve conduction velocities, endoneurial nutritive blood flow, vascular reactivity of epineurial arterioles, thermal nociception, tactile allodynia, and intraepidermal nerve fiber density. Advanced glycation end product and markers of oxidative stress, including nitrated protein levels in sciatic nerve, were evaluated by Western blot. Rats with 12-wk duration of diabetes developed motor and sensory nerve conduction deficits, thermal hypoalgesia, tactile allodynia, and intraepidermal nerve fiber loss. All these changes, including impairment of nerve blood flow and vascular reactivity of epineurial arterioles, were partially reversed by 4 wk of cariporide treatment. Na(+)/H(+) exchanger 1 inhibition was also associated with reduction of diabetes-induced accumulation of advanced glycation endproduct, oxidative stress, and nitrated proteins in sciatic nerve. In conclusion, these findings support an important role for Na(+)/H(+) exchanger 1 in functional, structural, and biochemical manifestations of peripheral diabetic neuropathy and provide the rationale for development of Na(+)/H(+) exchanger 1 inhibitors for treatment of diabetic vascular and neural complications.

Different roles of 12/15-lipoxygenase in diabetic large and small fiber peripheral and autonomic neuropathies.

Up-regulation of 12/15-lipoxygenase, which converts arachidonic acid to 12(S)- and 15(S)-hydroxyeicosatetraenoic acids, causes impaired cell signaling, oxidative-nitrosative stress, and inflammation. This study evaluated the role for 12/15-lipoxygenase in diabetic large and small fiber peripheral and autonomic neuropathies. Control and streptozotocin-diabetic wild-type and 12/15-lipoxygenase-deficient mice were maintained for 14 to 16 weeks. 12/15-lipoxygenase gene deficiency did not affect weight gain or blood glucose concentrations. Diabetic wild-type mice displayed increased sciatic nerve 12/15-lipoxygenase and 12(S)-hydroxyeicosatetraenoic acid levels. 12/15-lipoxygenase deficiency prevented or alleviated diabetes-induced thermal hypoalgesia, tactile allodynia, motor and sensory nerve conduction velocity deficits, and reduction in tibial nerve myelinated fiber diameter, but not intraepidermal nerve fiber loss. The frequencies of superior mesenteric-celiac ganglion neuritic dystrophy, the hallmark of diabetic autonomic neuropathy in mouse prevertebral sympathetic ganglia, were increased 14.8-fold and 17.2-fold in diabetic wild-type and 12/15-lipoxygenase-deficient mice, respectively. In addition, both diabetic groups displayed small (<1%) numbers of degenerating sympathetic neurons. In conclusion, whereas 12/15-lipoxygenase up-regulation provides an important contribution to functional changes characteristic for both large and small fiber peripheral diabetic neuropathies and axonal atrophy of large myelinated fibers, its role in small sensory nerve fiber degeneration and neuritic dystrophy and neuronal degeneration characteristic for diabetic autonomic neuropathy is minor. This should be considered in the selection of endpoints for future clinical trials of 12/15-lipoxygenase inhibitors.

Role of 12/15-lipoxygenase in nitrosative stress and peripheral prediabetic and diabetic neuropathies.

This study evaluated the role of 12/15-lipoxygenase, which converts arachidonic acid to 12(S)- and 15(S)-hydroxyeicosatetraenoic acids, in nitrosative stress in the peripheral nervous system and peripheral prediabetic and diabetic neuropathies. The experiments were performed in C57BL6/J mice made diabetic with streptozotocin or fed a high-fat diet and in human Schwann cells cultured in 5.5 or 30 mM glucose. 12/15-Lipoxygenase overexpression and activation were present in sciatic nerve and spinal cord of diabetic and high-fat diet-fed mice, as well as in human Schwann cells cultured in high concentrations of D-, but not L-glucose. 12/15-Lipoxygenase inhibition by cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (8 mg kg(-1) day(-1) sc, for 4 weeks after 12 weeks without treatment) alleviated the accumulation of nitrated proteins in the sciatic nerve and spinal cord, and large and small nerve fiber dysfunction, but not intraepidermal nerve fiber loss. 12/15-Lipoxygenase gene deficiency alleviated nitrosative stress and nerve conduction deficit, but not small sensory fiber neuropathy, in high-fat diet-fed mice. In conclusion, 12/15-lipoxygenase is implicated in nitrosative stress and peripheral neuropathy in mouse models of type 1 and early type 2 diabetes. Its presence in human Schwann cells and upregulation by high glucose suggest a potential involvement in human disease.

New therapeutic and biomarker discovery for peripheral diabetic neuropathy: PARP inhibitor, nitrotyrosine, and tumor necrosis factor-{alpha}.

This study evaluated poly(ADP-ribose) polymerase (PARP) inhibition as a new therapeutic approach for peripheral diabetic neuropathy using clinically relevant animal model and endpoints, and nitrotyrosine (NT), TNF-alpha, and nitrite/nitrate as potential biomarkers of the disease. Control and streptozotocin-diabetic rats were maintained with or without treatment with orally active PARP inhibitor 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI-15,427), 30 mg kg(-1) d(-1), for 10 wk after first 2 wk without treatment. Therapeutic efficacy was evaluated by poly(ADP-ribosyl)ated protein expression (Western blot analysis), motor and sensory nerve conduction velocities, and tibial nerve morphometry. Sciatic nerve and spinal cord NT, TNF-alpha, and nitrite/nitrate concentrations were measured by ELISA. NT localization in peripheral nervous system was evaluated by double-label fluorescent immunohistochemistry. A PARP inhibitor treatment counteracted diabetes-induced motor and sensory nerve conduction slowing, axonal atrophy of large myelinated fibers, and increase in sciatic nerve and spinal cord NT and TNF-alpha concentrations. Sciatic nerve NT and TNF-alpha concentrations inversely correlated with motor and sensory nerve conduction velocities and myelin thickness, whereas nitrite/nitrate concentrations were indistinguishable between control and diabetic groups. NT accumulation was identified in endothelial and Schwann cells of the peripheral nerve, neurons, astrocytes, and oligodendrocytes of the spinal cord, and neurons and glial cells of the dorsal root ganglia. The findings identify PARP as a compelling drug target for prevention and treatment of both functional and structural manifestations of peripheral diabetic neuropathy and provide rationale for detailed evaluation of NT and TNF-alpha as potential biomarkers of its presence, severity, and progression.

Peripheral neuropathy in mice with neuronal nitric oxide synthase gene deficiency.

Evidence for the important role of the potent oxidant peroxynitrite in peripheral diabetic neuropathy and neuropathic pain is emerging. This study evaluated the contribution of neuronal nitric oxide synthase (nNOS) to diabetes-induced nitrosative stress in peripheral nerve and dorsal root ganglia, and peripheral nerve dysfunction and degeneration. Control and nNOS-/- mice were made diabetic with streptozotocin, and maintained for 6 weeks. Peroxynitrite injury was assessed by nitrotyrosine and poly(ADP-ribose) immunoreactivities. Peripheral diabetic neuropathy was evaluated by measurements of sciatic motor and hind-limb digital sensory nerve conduction velocities, thermal algesia, tactile allodynia, and intraepidermal nerve fiber density. Control nNOS-/- mice displayed normal motor nerve conduction velocity and thermal response latency, whereas sensory nerve conduction velocity was slightly lower compared with non-diabetic wild-type mice, and tactile response threshold and intraepidermal nerve fiber density were reduced by 47 and 38%, respectively. Both diabetic wild-type and nNOS-/- mice displayed enhanced nitrosative stress in peripheral nerve. In contrast to diabetic wild-type mice, diabetic nNOS-/- mice had near normal nitrotyrosine and poly(ADP-ribose) immunofluorescence in dorsal root ganglia. Both diabetic wild-type and nNOS-/- mice developed motor and sensory nerve conduction velocity deficits and thermal hypoalgesia although nNOS gene deficiency slightly reduced severity of the three disorders. Tactile response thresholds were similarly decreased in control and diabetic nNOS-/- mice compared with non-diabetic wild-type mice. Intraepidermal nerve fiber density was lower by 27% in diabetic nNOS-/- mice compared with the corresponding non-diabetic group, and by 20% in diabetic nNOS-/- mice compared with diabetic wild-type mice. In conclusion, nNOS is required for maintaining the normal peripheral nerve function and small sensory nerve fibre innervation. nNOS gene deficiency does not protect from development of nerve conduction deficit, sensory neuropathy and intraepidermal nerve fiber loss.

PARP inhibition or gene deficiency counteracts intraepidermal nerve fiber loss and neuropathic pain in advanced diabetic neuropathy.

Evidence that poly(ADP-ribose) polymerase (PARP) activation plays an important role in diabetic complications is emerging. This study evaluated the role of PARP in rat and mouse models of advanced diabetic neuropathy. The orally active PARP inhibitor 10-(4-methylpiperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI-15427; formulated as a mesilate salt, 30 mg kg(-1) day(-1) in the drinking water for 10 weeks after the first 2 weeks without treatment) at least partially prevented PARP activation in peripheral nerve and DRG neurons, as well as thermal hypoalgesia, mechanical hyperalgesia, tactile allodynia, exaggerated response to formalin, and, most importantly, intraepidermal nerve fiber degeneration in streptozotocin-diabetic rats. These findings are consistent with the lack of small sensory nerve fiber dysfunction in diabetic PARP -/- mice. Furthermore, whereas diabetic PARP +/+ mice displayed approximately 46% intraepidermal nerve fiber loss, diabetic PARP -/- mice retained completely normal intraepidermal nerve fiber density. In conclusion, PARP activation is an important contributor to intraepidermal nerve fiber degeneration and functional changes associated with advanced Type 1 diabetic neuropathy. The results support a rationale for the development of potent and low-toxicity PARP inhibitors and PARP inhibitor-containing combination therapies.

Evaluation of the peroxynitrite decomposition catalyst Fe(III) tetra-mesitylporphyrin octasulfonate on peripheral neuropathy in a mouse model of type 1 diabetes.

Whereas the important role of free radicals in diabetes-associated complications is well established, the contributions of the highly reactive oxidant peroxynitrite have not been properly explored. The present study used a pharmacological approach to evaluate the role of peroxynitrite in peripheral diabetic neuropathy. Control and STZ-diabetic mice were maintained with or without treatment with the potent peroxynitrite decomposition catalyst Fe(III) tetramesitylporphyrin octasulfonate (FeTMPS), at doses of 5 or 10 mg/kg/day in the drinking water for 3 weeks after an initial 3 weeks without treatment. Mice with a 6-week duration of diabetes developed clearly manifest motor (MNCV) and sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia (paw withdrawal, tail-flick, and hot plate tests), mechanical hypoalgesia (tail pressure Randall-Sellito test), tactile allodynia (flexible von Frey filament test), and approximately 44% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, grey matter of the spinal cord, and dorsal root ganglion neurons. FeTMPS treatment alleviated or essentially corrected (at a dose of 10 mg/kg/day) MNCV and SNCV deficits, and was associated with less severe small sensory nerve fiber dysfunction and degeneration. Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons in peroxynitrite decomposition catalyst-treated diabetic mice was markedly reduced. In conclusion, peroxynitrite contributes to large motor, large sensory, and small sensory fiber neuropathy in streptozotocin-diabetic mice. The findings provide rationale for development of potent peroxynitrite decomposition catalysts for the treatment of diabetic neuropathy.

A peroxynitrite decomposition catalyst counteracts sensory neuropathy in streptozotocin-diabetic mice.

Whereas an important role of free radicals and oxidants in peripheral diabetic neuropathy is well established, the contribution of nitrosative stress and, in particular, of the highly reactive oxidant peroxynitrite, has not been properly explored. Our previous findings implicate peroxynitrite in diabetes-associated motor and sensory nerve conduction deficits and peripheral nerve energy deficiency and poly(ADP-ribose) polymerase activation associated with Type 1 diabetes. In this study the role of nitrosative stress in diabetic sensory neuropathy is evaluated. The peroxynitrite decomposition catalyst Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)pyridyl porphyrin (FP15) was administered to control and streptozotocin (STZ)-diabetic mice at the dose of 5 mg kg(-1) day(-1) (FP15), for 3 weeks after initial 3 weeks without treatment. Mice with 6-week duration of diabetes developed clearly manifest thermal hypoalgesia (paw withdrawal, tail-flick, and hot plate tests), mechanical hypoalgesia (tail pressure Randall-Sellito test), tactile allodynia (flexible von Frey filament test), and approximately 38% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, grey matter of spinal cord, and dorsal root ganglion neurons. FP15 treatment was associated with alleviation of thermal and mechanical hypoalgesia. Tactile response threshold tended to increase in response to peroxynitrite decomposition catalyst treatment, but still remained approximately 59% lower compared with non-diabetic controls. Intraepidermal nerve fiber density was 25% higher in FP15-treated than in untreated diabetic rats, but the difference between two groups did not achieve statistical significance (p=0.054). Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons of peroxynitrite decomposition catalyst-treated diabetic mice were markedly reduced. In conclusion, nitrosative stress plays an important role in sensory neuropathy associated with Type 1 diabetes. The findings provide rationale for further studies of peroxynitrite decomposition catalysts in a long-term diabetic model.

Nitrosative stress and peripheral diabetic neuropathy in leptin-deficient (ob/ob) mice.

Nitrosative stress contributes to nerve conduction slowing, thermal hypoalgesia, and impaired nitrergic innervation in animal models of Type 1 diabetes. The role for reactive nitrogen species in Type 2 diabetes-associated neuropathy remains unexplored. This study evaluated the role for nitrosative stress in functional and structural neuropathic changes in ob/ob mice, a model of Type 2 diabetes with mild hyperglycemia and obesity. Two structurally diverse peroxynitrite decomposition catalysts, Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)-pyridyl porphyrin (FP15) and Fe(III) tetra-mesitylporphyrin octasulfonate (FeTMPS), were administered to control and 8-week-old ob/ob mice for 3 weeks at the doses of 5 mg kg(-1) day(-1) (FP15) and 5 and 10 mg kg(-1) day(-1) (FeTMPS). The 11-week-old ob/ob mice developed motor nerve conduction velocity (MNCV) and hind-limb digital sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia, tactile allodynia, and a remarkable ( approximately 78%) loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, spinal cord, and dorsal root ganglion neurons. Treatment with two structurally diverse peroxynitrite decomposition catalysts was associated with restoration of normal MNCV and SNCV, and alleviation of thermal hypoalgesia. Tactile response thresholds increased in response to peroxynitrite decomposition catalyst treatment, but still remained approximately 2.7- to 3.2-fold lower compared with non-diabetic controls. Intraepidermal nerve fiber loss was not alleviated by either FP15 or FeTMPS. Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglia of peroxynitrite decomposition catalyst-treated ob/ob mice were essentially normal. In conclusion, nitrosative stress plays an important role in functional abnormalities associated with large motor, large sensory, and small sensory fiber neuropathy, but not in small sensory nerve fiber degeneration, in this animal model. Peroxynitrite decomposition catalysts alleviate Type 2 diabetes-associated sensory nerve dysfunction, likely by mechanism(s) not involving arrest of degenerative changes or enhanced regeneration of small sensory nerve fibers.

Morphometrical studies of reproductive system of birds after treatment with dopamine receptor blockers and melatonin.

Male Japanese quails were treated with melatonin alone or melatonin combined with D1 and D2 dopamine receptor blockers. Following the treatment, hypothalamus, pituitary glands and testes were analyzed morphometrically. The results suggest the existence of an interaction between melatonin and dopaminergic system in the brain in the regulation of reproductive processes in immature birds. The character of this interaction alters according to the time of the treatment (morning, afternoon, evening, night).