Female Dark Agouti Research Articles

Alterations in the Hypothalamic-Pituitary-Adrenal Axis as a Response to Experimental Autoimmune Encephalomyelitis in Dark Agouti Rats of Both Sexes.

Multiple sclerosis (MS) is a chronic inflammatory disease that affects the central nervous system, usually diagnosed during the reproductive period. Both MS and its commonly used animal model, experimental autoimmune encephalomyelitis (EAE), exhibit sex-specific features regarding disease progression and disturbances in the neuroendocrine and endocrine systems. This study investigates the hypothalamic-pituitary-adrenal (HPA) axis response of male and female Dark Agouti rats during EAE. At the onset of EAE, Crh expression in the hypothalamus of both sexes is decreased, while males show reduced plasma adrenocorticotropic hormone levels. Adrenal gland activity is increased during EAE in both males and females, as evidenced by enlarged adrenal glands and increased StAR gene and protein expression. However, only male rats show increased serum and adrenal corticosterone levels, and an increased volume of the adrenal cortex. Adrenal 3β-HSD protein and progesterone levels are elevated in males only. Serum progesterone levels of male rats are also increased, although testicular progesterone levels are decreased during the disease, implying that the adrenal gland is the source of elevated serum progesterone levels in males. Our results demonstrate a sex difference in the response of the HPA axis at the adrenal level, with male rats showing a more pronounced induction during EAE.

Sex Differences under Vitamin D Supplementation in an Animal Model of Progressive Multiple Sclerosis.

A central role for vitamin D (VD) in immune modulation has recently been recognized linking VD insufficiency to autoimmune disorders that commonly exhibit sex-associated differences. Similar to other autoimmune diseases, there is a higher incidence of multiple sclerosis (MS) in women, but a poorer prognosis in men, often characterized by a more rapid progression. Although sex hormones are most likely involved, this phenomenon is still poorly understood. Oxidative stress, modulated by VD serum levels as well as sex hormones, may act as a contributing factor to demyelination and axonal damage in both MS and the corresponding preclinical models. In this study, we analyzed sex-associated differences and VD effects utilizing an animal model that recapitulates histopathological features of the progressive MS phase (PMS). In contrast to relapsing-remitting MS (RRMS), PMS has been poorly investigated in this context. Male (n = 50) and female (n = 46) Dark Agouti rats received either VD (400 IU per week; VD+) or standard rodent food without extra VD (VD-) from weaning onwards. Myelination, microglial activation, apoptotic cell death and neuronal viability were assessed using immunohistochemical markers in brain tissue. Additionally, we also used two different histological markers against oxidized lipids along with colorimetric methods to measure protective polyphenols (PP) and total antioxidative capacity (TAC) in serum. Neurofilament light chain serum levels (sNfL) were analyzed using single-molecule array (SIMOA) analysis. We found significant differences between female and male animals. Female rats exhibited a better TAC and higher amounts of PP. Additionally, females showed higher myelin preservation, lower microglial activation and better neuronal survival while showing more apoptotic cells than male rats. We even found a delay in reaching the peak of the disease in females. Overall, both sexes benefitted from VD supplementation, represented by significantly less cortical, neuroaxonal and oxidative damage. Unexpectedly, male rats had an even higher overall benefit, most likely due to differences in oxidative capacity and defense systems.

Abdominal vagus nerve stimulation alleviates collagen-induced arthritis in rats.

Rheumatoid arthritis (RA) is a chronic, autoimmune inflammatory disease. Despite therapeutic advances, a significant proportion of RA patients are resistant to pharmacological treatment. Stimulation of the cervical vagus nerve is a promising alternative bioelectric neuromodulation therapeutic approach. However, recent clinical trials show cervical vagus nerve stimulation (VNS) was not effective in a significant proportion of drug resistant RA patients. Here we aim to assess if abdominal vagus nerve stimulation reduces disease severity in a collagen-induced arthritis (CIA) rat model. The abdominal vagus nerve of female Dark Agouti rats was implanted and CIA induced using collagen type II injection. VNS (1.6 mA, 200 μs pulse width, 50 μs interphase gap, 27 Hz frequency) was applied to awake freely moving rats for 3 h/day (days 11-17). At 17 days following the collagen injection, unstimulated CIA rats (n = 8) had significantly worse disease activity index, tumor necrosis factor-alpha (TNF-α) and receptor activator of NFκB ligand (RANKL) levels, synovitis and cartilage damage than normal rats (n = 8, Kruskal-Wallis: P < 0.05). However, stimulated CIA rats (n = 5-6) had significantly decreased inflammatory scores and ankle swelling (Kruskal-Wallis: P < 0.05) compared to unstimulated CIA rats (n = 8). Levels of tumor necrosis factor-alpha (TNF-α) remained at undetectable levels in stimulated CIA rats while levels of receptor activator of NFκB ligand (RANKL) were significantly less in stimulated CIA rats compared to unstimulated CIA rats (P < 0.05). Histopathological score of inflammation and cartilage loss in stimulated CIA rats were no different from that of normal (P > 0.05). In conclusion, abdominal VNS alleviates CIA and could be a promising therapy for patients with RA.

Prevention of Triglyceridemia by (Non-)Anticoagulant Heparin(oids) Does Not Preclude Transplant Vasculopathy and Glomerulosclerosis.

Background: In renal transplantation, chronic transplant dysfunction (CTD) is associated with increased PCSK9 and dyslipidemia. PCSK9 is an enzyme that increases plasma cholesterol levels by downregulating LDLR expression. We recently showed increased PCSK9–syndecan-1 interaction in conditions of proteinuria and renal function loss. Treatment with heparin(oids) might be a therapeutic option to improve dyslipidemia and CTD. We investigated the effects of (non-)anticoagulant heparin(oids) on serum lipids, syndecan-1 and PCSK9 levels, and CTD development. Methods: Kidney allotransplantation was performed from female Dark Agouti to male Wistar Furth recipients. Transplanted rats received daily subcutaneous injections of saline, unfractionated heparin, and RO-heparin or NAc-heparin (2 mg heparin(oid)/kg BW) until sacrifice after 9 weeks of treatment. Results: Saline-treated recipients developed hypertension, proteinuria, and loss of creatinine clearance (all p < 0.05 compared to baseline), along with glomerulosclerosis and arterial neo-intima formation. Saline-treated recipients showed significant increase in plasma triglycerides (p < 0.05), borderline increase in non-HDLc/HDLc (p = 0.051), and ∼10-fold increase in serum syndecan-1 (p < 0.05), without significant increase in serum PCSK9 at 8 weeks compared to baseline. Heparin and non-anticoagulant RO-heparin administration in transplanted rats completely prevented an increase in triglycerides compared to saline-treated recipients at 8 weeks (both p < 0.05). Heparin(oids) treatment did not influence serum total cholesterol (TC), plasma syndecan-1 and PCSK9 levels, creatinine clearance, proteinuria, glomerulosclerosis, and arterial neo-intima formation, 8 weeks after transplantation. Combining all groups, increased syndecan-1 shedding was associated with TC (r = 0.5; p = 0.03) and glomerulosclerosis (r = 0.53; p = 0.021), whereas the non-HDLc/HDLc ratio was associated with the neo-intimal score in the transplanted kidneys (r = 0.65; p < 0.001). Conclusion: Prevention of triglyceridemia by (non-)anticoagulant heparin(oids) neither influenced PCSK9/syndecan-1 nor precluded CTD, which however did associate with the shedding of lipoprotein clearance receptor syndecan-1 and the unfavorable cholesterol profile.

Dose-dependent effect of myelin oligodendrocyte glycoprotein on visual function and optic nerve damage in experimental autoimmune encephalomyelitis.

Female Dark Agouti rats were immunized with increasing doses of myelin oligodendrocyte glycoprotein (MOG) to develop experimental autoimmune encephalomyelitis (EAE), a preclinical model of multiple sclerosis. Typical EAE motor impairments were assessed daily and noninvasive visual evoked potentials (VEPs) were recorded at baseline and 5weeks after immunization, with final histopathology of optic nerves (ONs). Immunized rats exhibited a relapsing-remitting clinical course. Both VEP and histological abnormalities were detected in a MOG dose-dependent gradient. Increasing MOG dosage augmented visual function impairment in EAE, which could be monitored with VEP recording to assess demyelination and axonal loss along ONs.

Cytochrome P450 2D (CYP2D) enzyme dysfunction associated with aging and serotonin deficiency in the brain and liver of female Dark Agouti rats

Among the enzymes that support brain metabolism, cytochrome P450 (CYP) enzymes occupy an important place. These enzymes catalyze the biotransformation pathways of neuroactive endogenous substrates (neurosteroids, neurotransmitters) and are necessary for the detoxification processes. The aim of the present study was to assess changes in the CYP2D activity and protein level during the aging process and as a result of serotonin deficiency in the female brain. The CYP2D activity was measured in brain and liver microsomes of Dark Agouti wild type (WT) female rats (mature 15-week-old and senescent 18-month-old rats) and in tryptophan hydroxylase 2 (TPH2)-deficient senescent female rats. The CYP2D activity in mature WT Dark Agouti females was independent of the changing phases of the estrous cycle. In senescent WT females rats, the CYP2D activity and protein level were decreased in the cerebral cortex, hippocampus, cerebellum and liver, but increased in the brain stem. In the other examined structures (frontal cortex, hypothalamus, thalamus, striatum), the enzyme activity did not change. In aging TPH2-deficient females, the CYP2D activity and protein levels were decreased in the frontal cortex, hypothalamus and brain stem (activity only), remaining unchanged in other brain structures and liver, relative to senescent WT females. In summary, the aging process and TPH2 deficit affect the CYP2D activity and protein level in female rats, which may have a negative impact on the compensatory capacity of CYP2D in the synthesis of serotonin and dopamine in cerebral structures involved in cognitive and emotional functions. In the liver, the CYP2D-catalyzed drug metabolism may be diminished in elderly females. The results in female rats are compared with those obtained previously in males. It is concluded that aging and serotonin deficiency exert sex-dependent effects on brain CYP2D, which seem to be less favorable in females concerning CYP2D-mediated neurotransmitter synthesis, but beneficial regarding slower neurosteroid metabolism.

Neuroimmunological complications arising from chemotherapy-induced gut toxicity and opioid exposure in female dark agouti rats.

Cancer patients may experience symptom clusters, including chemotherapy-induced (CI) gut toxicity (CIGT) and cognitive impairment. Analgesic selection for pain associated with CIGT is difficult as opioids induce glial reactivity and unwanted side effects. This study quantified central glial reactivity and proinflammatory effects in rats with CIGT using three mechanistically different analgesics. Regional adaptations were indicative of immune-to-brain signaling routes. Utilizing a 5-fluorouracil-induced GT (5IGT) rat model and analgesic intervention (carprofen (CAR), buprenorphine (BUP), and tramadol (TRAM)), spinal and brain neuroimmune modulation was examined via microglial, astrocyte, and proinflammatory (cluster of differentiation molecule 11b; CD11b, glial fibrillary associated protein; GFAP, and interleukin-1 beta; IL1β) reactivity marker expression changes by western blot analysis. 5IGT significantly increased thoracic GFAP (p<0.05) and IL-1β (p<0.0001) expression, CAR and BUP ameliorated these effects. BUP and TRAM with 5-FU synergistically increased hippocampal GFAP expression. CAR administered with 5IGT significantly elevated hippocampal and thoracic CD11b expression levels (p<0.05). The neuroimmune responses observed in this study suggest activation of peripheral-to-central immune signaling pathways. We speculate that the opioid-induced hippocampal changes inferred a humorally mediated mechanism, whereas thoracic neuroimmune modifications indicated activation of an indirect neural route. Although TRAM ameliorated 5IGT-intestinal inflammation, this opioid presents complications relating to bodyweight and regional glial dysregulation (neuroinflammation) and may not be optimal in the management of pain associated with 5IGT. The chemotherapy-induced gut-derived neuroimmune consequences observed suggest a potential mechanistic contribution to central components of the cancer symptom cluster experience, while the opioid-related glial changes have implications for optimal pain management in this setting warranting further investigation.

Auditory brainstem responses in aging dark agouti rats.

The present study examined auditory function across age in the dark agouti (DA) rat strain. Auditory brainstem responses (ABRs) were measured for frequencies 8, 16, and 32 kHz in male and female DA rats from 3 to 18 months of age. Hearing thresholds and absolute and interpeak latencies (IPLs) were analyzed. Male hearing thresholds remained stable for the first year of life and then significantly increased at 18 months across all frequencies; female hearing remained stable at all tested ages out to 18 months. At 12 months, male DA rats showed significantly longer absolute latencies by age (i.e., compared with 3-month-old males) and sex (compared with 12-month-old females), with no differences in IPLs. At 18 months, female DA rats showed significantly longer absolute latencies with age (compared with 3-month-old females) and sex (compared with 18-month-old males), particularly for the later waves. Female IPLs were also significantly longer with age and by sex for the later waves. This report supports the feasibility of using male DA rats in studies to investigate age-related hearing loss (ARHL; presbycusis).

Sex differences in Tfh cell help to B cells contribute to sexual dimorphism in severity of rat collagen-induced arthritis

The study examined germinal centre (GC) reaction in lymph nodes draining inflamed joints and adjacent tissues (dLNs) in male and female Dark Agouti rat collagen type II (CII)-induced arthritis (CIA) model of rheumatoid arthritis. Female rats exhibiting the greater susceptibility to CIA mounted stronger serum CII-specific IgG response than their male counterparts. This correlated with the higher frequency of GC B cells in female compared with male dLNs. Consistently, the frequency of activated/proliferating Ki-67+ cells among dLN B cells was higher in females than in males. This correlated with the shift in dLN T follicular regulatory (Tfr)/T follicular helper (Tfh) cell ratio towards Tfh cells in females, and greater densities of CD40L and CD40 on their dLN T and B cells, respectively. The higher Tfh cell frequency in females was consistent with the greater dLN expression of mRNA for IL-21/27, the key cytokines involved in Tfh cell generation and their help to B cells. Additionally, in CII-stimulated female rat dLN cell cultures IFN-γ/IL-4 production ratio was shifted towards IFN-γ. Consistently, the serum IgG2a(b)/IgG1 CII-specific antibody ratio was shifted towards an IgG2a(b) response in females. Thus, targeting T-/B-cell interactions should be considered in putative further sex-based translational pharmacology research.

Prebiotics Fructo-, Galacto-, and Mannan-Oligosaccharide Do Not Protect against 5-Fluorouracil–Induced Intestinal Mucositis in Rats

Prebiotics selectively stimulate the growth of beneficial bacteria within the gastrointestinal tract, and have been investigated in human and animal studies for their capacity to improve intestinal health. We investigated the prebiotics fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), and mannan-oligosaccharide (MOS) for their potential to alleviate intestinal damage in rats. Female Dark Agouti rats (6-8 wk old, 110-150 g) were allocated to 1 of the following treatment groups (n=8/group): saline/water, saline/FOS, saline/GOS, saline/MOS, 5-fluorouracil (5FU)/water, 5FU/FOS, 5FU/GOS, and 5FU/MOS. Rats were pretreated with either 5% GOS, MOS, or FOS or vehicle (water) from day -12 to day 0. On day 0, rats received a single intraperitoneal injection of saline or 5FU. Metabolic data were recorded daily and all rats were killed on day 3. Histopathology was quantified in hematoxylin and eosin-stained sections. Intestinal sucrase and myeloperoxidase activity were quantified by biochemical assay. Fecal SCFAs-acetic, propionic, and butyric acid-were also measured. Statistical analysis was by repeated-measures, 2-factor ANOVA or Kruskal-Wallis and Mann-Whitney U test; P<0.05 was considered statistically significant. Body weight was significantly decreased in all treatment groups after 5FU injection, with no change in body weight observed in any prebiotic treatment group. Total food intake was lower by ≥7% in the GOS treatment group pre-5FU than in all other groups (P<0.05). Ileal villus height was 18% higher in GOS-treated rats pre-5FU than in respective water controls (P<0.05). Jejunal and ileal villus height and crypt depth were significantly decreased in all treatment groups after 5FU injection, with no prebiotic effect observed. SCFAs were differentially increased in prebiotic treatment groups compared with water-only controls (P<0.05). FOS, GOS, and MOS have differential effects in modifying small intestinal pathology and SCFA profiles in rats with healthy and damaged small intestinal mucosa.

Use of the Rat Grimace Scale to Evaluate Visceral Pain in a Model of Chemotherapy-Induced Mucositis.

Simple SummaryMucositis is a painful and often debilitating condition associated with cancer treatment. Management of associated symptoms is an important clinical consideration. Animal models are used in mucositis research to model the condition in humans in order to develop novel therapeutic agents to relieve symptoms. Previous animal studies have focused on disease severity and outcomes, but often failed to measure pain. The rat grimace scale (RGS) is a validated observational measure used to gauge pain levels experienced by rats. The aim of this study was to assess the rat grimace scale in a rat model of mucositis, and to examine whether changes in clinical signs and anxiety reflected the grimace responses recorded. We also aimed to determine whether the responses were pain-specific by administering potent opioid painkilling agents. In the present study rat grimace scores did not change significantly between treatments. Development of reliable pain assessment methods in animal models is urgently required to improve model relevance to human clinical practice, in addition to safeguarding animal welfare. The rat grimace scale (RGS) is a measure of spontaneous pain that evaluates pain response. The ability to characterize pain through a non-invasive method has considerable utility for numerous animal models of disease, including mucositis, a painful, self-limiting side-effect of chemotherapy treatment. Preclinical studies investigating novel therapeutics for mucositis often focus on pathological outcomes and disease severity. These investigations fail to measure pain, in spite of reduction of pain being a key clinical therapeutic goal. This study assessed the utility of the RGS for pain assessment in a rat model of mucositis, and whether changes in disease activity index (DAI) and open field test (OFT) reflected the grimace responses recorded. Sixty tumor-bearing female Dark Agouti rats were injected with either saline or 5-Fluourouracil alone, or with co-administration of opioid analgesics. Whilst differences in DAI were observed between treatment groups, no difference in RGS scores or OFT were demonstrated. Significant increases in grimace scores were observed across time. However, whilst a statistically significant change may have been noted, the biological relevance is questionable in terms of practical usage, since an observer is only able to score whole numbers. Development of effective pain assessment methods in animal models is required to improve welfare, satisfy regulatory requirements, and increase translational validity of the model to human patients.

Selective MMP Inhibition, Using AZD3342, to Reduce Gastrointestinal Toxicity and Enhance Chemoefficacy in a Rat Model

Background: The common cytotoxic mechanisms that underpin chemoefficacy and toxicity have hampered efforts to deliver effective supportive care interventions, particularly for gastrointestinal (GI) toxicity. Matrix metalloproteinases (MMPs) have been implicated in both tumor growth and GI toxicity, and as such MMP inhibitors present as a novel therapeutic avenue to simultaneously enhance treatment efficacy and reduce toxicity. Objectives: The aim of this study was to determine the efficacy of an MMP-9/12 inhibitor, AZD3342, on tumor growth and GI toxicity in a rat model. Methods: Female tumor-bearing Dark Agouti rats (n = 90) were divided into 4 groups: vehicle control; methotrexate (MTX); AZD3342, and MTX + AZD3342. Tumors were measured daily (for 5 days) using digital calipers. GI toxicity was assessed using well-established clinical markers (diarrhea/weight loss), histopathological analysis, and functional assessment of intestinal barrier permeability. Results: AZD3342 delayed the onset of severe diarrhea by 1 day (vs. MTX) but was unable to improve the overall severity of diarrhea. No changes were detected in tissue morphology or intestinal barrier function. AZD3342 alone suppressed tumor growth (p = 0.003 vs. vehicle) but did not enhance the efficacy of MTX. Conclusions: This study showed partial efficacy of AZD3342 in reducing tumor growth and delaying the onset of severe diarrhea caused by MTX in rats. We suggest further studies be undertaken targeting appropriate scheduling of AZD3342 as well as investigating different cytotoxic therapies that strongly activate MMP signaling.

Collagen-induced arthritis in Dark Agouti rats as a model for study of immunological sexual dimorphisms in the human disease

Collagen-induced arthritis (CIA) is a frequently used animal model of rheumatoid arthritis, human autoimmune disease that exhibits clear sex bias in incidence and clinical course. Female Dark Agouti rats immunized for CIA showed also greater incidence and higher arthritic score than their male counterparts. The study investigated sex differences in mechanisms controlling the primary immune responses in draining lymph nodes (dLNs), as a factor contributing to this dimorphism. The higher frequencies of CD4 + CD25 + Foxp3- cells, presumably activated effector T (Teff) cells, and IL-17+, IFN-γ + and IL-17 + IFN-γ + T cells were found in female compared with male rat dLNs. However, the frequency of CD4 + CD25 + Foxp3+ T regulatory cells (Treg) did not differ between sexes. Thus, CD4+ Teff cells/Treg ratio, and IL-17+ T cells/Treg and IFN-γ + T cells/Treg ratios were higher in female than in male rats, and among them was found lower frequency of PD-1+ cells. This suggested less efficient control of (auto)immune Th1/Th17 cell responses in female rat dLNs. On the contrary, the frequency of IL-4+ T cells was lower in female than in male rat dLNs. Consistently, the ratio of serum levels of collagen-specific IgG2a (IFN-γ-dependent, with an important pathogenic role in CIA) and IgG1 (IL-4-dependent) was shifted towards IgG2a in female compared with male rats. As a whole, the study suggests that sexual dimorphism in the control of T cell activation/polarization could contribute to sex bias in the susceptibility to CIA. Moreover, the study advises the use of animals of both sexes in the preclinical testing of new drugs for rheumatoid arthritis.

Voltage Gated Potassium Channel Kv1.3 Is Upregulated on Activated Astrocytes in Experimental Autoimmune Encephalomyelitis.

Kv1.3 is a voltage gated potassium channel that has been implicated in pathophysiology of multiple sclerosis (MS). In the present study we investigated temporal and cellular expression pattern of this channel in the lumbar part of spinal cords of animals with experimental autoimmune encephalomyelitis (EAE), animal model of MS. EAE was actively induced in female Dark Agouti rats. Expression of Kv1.3 was analyzed at different time points of disease progression, at the onset, peak and end of EAE. We here show that Kv1.3 increased by several folds at the peak of EAE at both gene and protein level. Double immunofluorescence analyses demonstrated localization of Kv1.3 on activated microglia, macrophages, and reactive astrocytes around inflammatory lesions. In vitro experiments showed that pharmacological block of Kv1.3 in activated astrocytes suppresses the expression of proinflammatory mediators, suggesting a role of this channel in inflammation. Our results support the hypothesis that Kv1.3 may be a therapeutic target of interest for MS and add astrocytes to the list of cells whose activation would be suppressed by inhibiting Kv1.3 in inflammatory conditions.

Effects of commercially produced almond by-products on chemotherapy-induced mucositis in rats.

AIMTo determine if almond extracts reduce the severity of chemotherapy-induced mucositis as determined through biochemical, histological and behavioural markers.METHODSIntestinal mucositis is a debilitating condition characterized by inflammation and ulceration of the gastrointestinal mucosa experienced by cancer patients undergoing chemotherapy. Certain bioactive plant products have shown promise in accelerating mucosal repair and alleviating clinical symptoms. This study evaluated almond extracts for their potential to reduce the severity of chemotherapy-induced mucositis in Dark Agouti rats. Female Dark Agouti rats were gavaged (days 3-11) with either PBS, almond hull or almond blanched water extract at two doses, and were injected intraperitoneally with 5-fluorouracil (5-FU-150 mg/kg) or saline on day 9 to induce mucositis. Burrowing behavior, histological parameters and myeloperoxidase activity were assessed.RESULTSBodyweight was significantly reduced in rats that received 5-FU compared to saline-treated controls (P < 0.05). Rats administered 5-FU significantly increased jejunal and ileal MPO levels (1048%; P < 0.001 and 409%; P < 0.001), compared to healthy controls. Almond hull extract caused a pro-inflammatory response in rats with mucositis as evidenced by increased myeloperoxidase activity in the jejunum when compared to 5-FU alone (rise 50%, 1088 ± 96 U/g vs 723 ± 135 U/g, P = 0.02). Other extract-related effects on inflammatory activity were minimal. 5-FU significantly increased histological severity score compared to healthy controls confirming the presence of mucositis (median of 9.75 vs 0; P < 0.001). The extracts had no ameliorating effect on histological severity score in the jejunum or ileum. Burrowing behavior was significantly reduced in all chemotherapy-treated groups (P = 0.001). The extracts failed to normalize burrowing activity to baseline levels.CONCLUSIONAlmond extracts at these dosages offer little beneficial effect on mucositis severity. Burrowing provides a novel measure of affective state in studies of chemotherapy-induced mucositis.

Increased migration of antigen presenting cells to newly-formed lymphatic vessels in transplanted kidneys by glycol-split heparin.

BackgroundChronic renal transplant dysfunction is characterized by loss of renal function and tissue remodeling, including chronic inflammation and lymph vessel formation. Proteoglycans are known for their chemokine presenting capacity. We hypothesize that interruption of the lymphatic chemokine–proteoglycan interaction interferes with the lymphatic outflow of leukocytes from the renal graft and might decrease the anti-graft allo-immune response.MethodsIn a rat renal chronic transplant dysfunction model (female Dark-Agouti to male Wistar Furth), chemokines were profiled by qRT-PCR in microdissected tubulo-interstitial tissue. Disruption of lymphatic chemokine–proteoglycan interaction was studied by (non-anticoagulant) heparin-derived polysaccharides in vitro and in renal allografts. The renal allograft function was assessed by rise in plasma creatinine and urea.ResultsWithin newly-formed lymph vessels of transplanted kidneys, numerous CD45+ leukocytes were found, mainly MHCII+, ED-1-, IDO-, HIS14-, CD103- antigen presenting cells, most likely representing a subset of dendritic cells. Treatment of transplanted rats with regular heparin and two different (non-)anticoagulant heparin derivatives revealed worsening of kidney function only in the glycol-split heparin treated group despite a two-fold reduction of tubulo-interstitial leukocytes (p<0.02). Quantitative digital image analysis however revealed increased numbers of intra-lymphatic antigen-presenting cells only in the glycol-split heparin group (p<0.01). The number of intra-lymphatic leukocytes significantly correlates with plasma creatinine and urea, and inversely with creatinine clearance.ConclusionsTreatment of transplanted rats with glycol-split heparin significantly increases the number of intra-lymphatic antigen presenting cells, by increased renal diffusion of lymphatic chemokines, thereby increasing the activation and recruitment of antigen presenting cells towards the lymph vessel. This effect is unwanted in the transplantation setting, but might be advantageous in e.g., dendritic cell vaccination.

Fractionated abdominal irradiation induces intestinal microvascular changes in an in vivo model of radiotherapy-induced gut toxicity.

Radiotherapy-induced gut toxicity (RIGT) is associated with diarrhoea, pain and rectal bleeding and can occur as an acute or chronic toxicity. The microvasculature has been shown to be altered in the development of RIGT; however, the features are not yet characterized. We hypothesized that apoptosis of microvascular cells would occur early in the gastrointestinal tract following fractionated irradiation, followed by late microvascular changes, including sclerosis and telangiectasis. Female Dark Agouti rats were treated with a 6-week fractionated radiation schedule of 3×2.5Gy doses per week localized to the abdomen. At 3, 6 and 15weeks, the intestines were assessed for markers of acute and chronic injury including morphological changes, collagen deposition, apoptosis and proliferation. Apoptosis of microvascular cells significantly increased at 6 and 15weeks in the jejunum (p=0.0026 and p=0.0062, respectively) and at 6 and 15weeks in the colon (p<0.0001 and p=0.0005, respectively) in rats receiving fractionated radiation to the abdomen. Histopathological changes of the colon microvasculature were also seen from week 3, including thickening of the lamina propria and dilated, thickened, telangiectatic vessels. Findings of this study provide evidence of regional and timing-specific changes in the intestinal microvasculature in response to fractionated radiotherapy which may play a role in development of both acute and chronic RIGT.

Effects of Supernatants from Escherichia coli Nissle 1917 and Faecalibacterium prausnitzii on Intestinal Epithelial Cells and a Rat Model of 5-Fluorouracil-Induced Mucositis

ABSTRACTFaecalibacterium prausnitzii (Fp) and Escherichia coli Nissle 1917 (EcN) are probiotics, which have been reported to ameliorate certain gastrointestinal disorders. We evaluated the effects of supernatants (SN) derived from Fp and EcN on 5-fluorouracil (5-FU)-treated intestinal cells and in a rat model of mucositis. In vitro: IEC-6, Caco-2, and T-84 cells were analyzed for viability and monolayer permeability. In vivo: Female dark agouti rats were gavaged with Fp or EcN SN and injected intraperitoneally with saline (control) or 5-FU to induce mucositis. Rats were euthanized and intestinal tissues collected for myeloperoxidase assay and histological analyses. In vitro: Caco-2 cell viability was further reduced when treated with Fp SN + 5-FU compared to 5-FU controls. In both Caco-2 and T-84 cells, Fp SN partially prevented the decrease in transepithelial electrical resistance (TER) caused by 5-FU administration. In vivo: 5-FU-injected rats administered Fp SN or EcN SN partly prevented body weight loss and normalized water intake compared to 5-FU controls. These results suggest a growth inhibitory mechanism of Fp SN action on transformed epithelial cells that could be mediated by effects on tight junctions. Factors derived from Fp SN and EcN SN could have a role in reducing the severity of intestinal mucositis.

Potential safety concerns of TLR4 antagonism with irinotecan: a preclinical observational report.

Irinotecan-induced gut toxicity is mediated in part by Toll-Like receptor 4 (TLR4) signalling. The primary purpose of this preclinical study was to determine whether blocking TLR4 signalling by administering (-)-naloxone, a TLR4 antagonist, would improve irinotecan-induced gut toxicity. Our secondary aim was to determine the impact of (-)-naloxone on tumour growth. Female Dark Agouti (DA) tumour-bearing rats were randomly assigned to four treatments (n=6 in each); control, (-)-naloxone (100mg/kg oral gavage at -2, 24, 48, and 72h), irinotecan (175mg/kg intraperitoneal at 0h), and (-)-naloxone and irinotecan. Body weight and tumour growth were measured daily, and diarrhoea incidence and severity were recorded 4× per day up to 72h post-treatment. At 72h, all rats that received irinotecan lost weight compared to controls (p=0.03). In addition, rats that received (-)-naloxone and irinotecan lost significantly more weight compared to controls (p<0.005) than irinotecan only compared to controls (p=0.001). (-)-Naloxone did not attenuate irinotecan-induced severe diarrhoea at 48 and 72h. Finally, (-)-naloxone caused increased tumour growth compared to control at 72h (p<0.05) and significantly reduced the efficacy of irinotecan (p=0.001). (-)-Naloxone in our preclinical model was unable to block irinotecan-induced gut toxicity and decreased the efficacy of irinotecan. As (-)-naloxone-oxycodone combination is used for cancer pain, this may present a potential safety concern for patients receiving (-)-naloxone-oxycodone and irinotecan concurrently and requires further investigation.

Long-Term Effects of (-)-Epigallocatechin Gallate (EGCG) on Pristane-Induced Arthritis (PIA) in Female Dark Agouti Rats.

Rheumatoid arthritis (RA)—a widespread chronic inflammatory disease in industrialized countries—is characterized by a persistent and progressive joint destruction. The chronic pro-inflammatory state results from a mutual activation of the innate and the adaptive immune system, while the exact pathogenesis mechanism is still under discussion. New data suggest a role of the innate immune system and especially polymorphonuclear granulocytes (PMNs, neutrophils) not only during onset and the destructive phase of RA but also at the chronification of the disease. Thereby the enzymatic activity of myeloperoxidase (MPO), a peroxidase strongly abundant in neutrophils, may be important: While its peroxidase activity is known to contribute to cartilage destruction at later stages of RA the almost MPO-specific oxidant hypochlorous acid (HOCl) is also discussed for certain anti-inflammatory effects. In this study we used pristane-induced arthritis (PIA) in Dark Agouti rats as a model for the chronic course of RA in man. We were able to shown that a specific detection of the HOCl-producing MPO activity provides a sensitive new marker to evaluate the actual systemic inflammatory status which is only partially detectable by the evaluation of clinical symptoms (joint swelling and redness measurements). Moreover, we evaluated the long-term pharmacological effect of the well-known anti-inflammatory flavonoid epigallocatechin gallate (EGCG). Thereby only upon early and continuous oral application of this polyphenol the arthritic symptoms were considerably diminished both in the acute and in the chronic phase of the disease. The obtained results were comparable to the treatment control (application of methotrexate, MTX). As revealed by stopped-flow kinetic measurements, EGCG may regenerate the HOCl-production of MPO which is known to be impaired at chronic inflammatory diseases like RA. It can be speculated that this MPO activity-promoting effect of EGCG may contribute to the pharmacological mode of action of this polyphenol.