Quantitative Histological Examination Research Articles

The neuropathological basis of elevated serum neurofilament light following experimental concussion

Mild traumatic brain injury (mTBI) or concussion is a substantial health problem globally, with up to 15% of patients experiencing persisting symptoms that can significantly impact quality of life. Currently, the diagnosis of mTBI relies on clinical presentation with ancillary neuroimaging to exclude more severe forms of injury. However, identifying patients at risk for a poor outcome or protracted recovery is challenging, in part due to the lack of early objective tests that reflect the relevant underlying pathology. While the pathophysiology of mTBI is poorly understood, axonal damage caused by rotational forces is now recognized as an important consequence of injury. Moreover, serum measurement of the neurofilament light (NfL) protein has emerged as a potentially promising biomarker of injury. Understanding the pathological processes that determine serum NfL dynamics over time, and the ability of NfL to reflect underlying pathology will be critical for future clinical research aimed at reducing the burden of disability after mild TBI. Using a gyrencephalic model of head rotational acceleration scaled to human concussion, we demonstrate significant elevations in serum NfL, with a peak at 3 days post-injury. Moreover, increased serum NfL was detectable out to 2 weeks post-injury, with some evidence it follows a biphasic course. Subsequent quantitative histological examinations demonstrate that axonal pathology, including in the absence of neuronal somatic degeneration, was the likely source of elevated serum NfL. However, the extent of axonal pathology quantified via multiple markers did not correlate strongly with the extent of serum NfL. Interestingly, the extent of blood–brain barrier (BBB) permeability offered more robust correlations with serum NfL measured at multiple time points, suggesting BBB disruption is an important determinant of serum biomarker dynamics after mTBI. These data provide novel insights to the temporal course and pathological basis of serum NfL measurements that inform its utility as a biomarker in mTBI.

Impact of Suturing Techniques on Microvascular Anastomosis Maturation.

INTRODUCTION Microvascular anastomosis using interrupted suture is a widely accepted standard technique. Continuous suture is less common due to the presumption that its firmness can negatively affect anastomosis maturation. The purpose of this study was to determine whether the use of continuous suture allows maturation of the microanastomosis site. MATERIAL AND METHODS A rat common carotid artery (CCA) end-to-end microanastomosis model was utilized, with 19 Long-Evans rats in the interrupted sutures group and 13 in the continuous suture group. Immediate blood flow of the operated and contralateral intact CCAs was compared before clamping, at the completion of the anastomosis and after 14 days. Quantitative transit time flowmetry measurement and histological examination were employed. RESULTS Initial blood flow in both intact CCAs was similar across all animals (p = .004). In the interrupted suture group, median anastomosis blood flow was 88.9% of the contralateral CCA blod flow, with a median suture time of 46 minutes. After two weeks, blood flow increased to 96.1%. In the continuous suture group, median anastomosis blood flow was 88.3% of the contralateral CCA blood flow, with a median suture time of 30 minutes. After two weeks, blood flow increased to 100.0%. The reduction in suture time achieved with continuous suture was 34.8% (p < .001). Histological examination confirmed scar maturity. CONCLUSIONS The maturation rates of continuous and interrupted suture microanastomosis were comparable in our study, implying that concerns about the suture restricting maturation may be unwarranted. Additional finding is the potential for a reduction in microanastomosis time when using the continuous suture technique.

256 The Neuropathological Basis of Elevated Serum Neurofilament Light Following Experimental Concussion

INTRODUCTION: Mild traumatic brain injury (mTBI), or concussion, is a substantial health problem, with up to 15% of patients experiencing persisting symptoms. Identifying patients at risk for a poor outcome or protracted recovery remains challenging. Axonal damage caused by rotational forces is now recognized as an important consequence of injury. Moreover, serum measurement of the neurofilament light (NfL) protein has emerged as a potentially promising biomarker of injury. METHODS: A gyrencephalic, porcine model of head rotational acceleration scaled to human mTBI was performed with survival of 48 hr (n = 2), 72 hr (n = 6) and 14 days (n = 4) and compared with shams at 72 hr (n = 1) and 14 days (n = 1). Serum NfL was measured pre-injury and at 40 min, 1.5 hr, 3-4 hr, 8 hr, 18 hr, 24 hr, 2 days, 3 days and 14 days post-injury. Detailed neuropathological assessment was performed including staining for markers of cell death (Fluoro-Jade-C), axonal injury (amyloid precursor protein and NfL), and blood-brain barrier (BBB) disruption (fibrinogen). RESULTS: Significant elevations in serum-NfL were identified following mTBI, with a peak at 72hr post-injury. Moreover, increased serum-NfL was detectable out to 2 weeks post-injury. Subsequent quantitative histological examinations demonstrated that axonal pathology was the likely source of elevated serum-NfL. However, the extent of axonal pathology quantified via multiple markers did not correlate strongly with the extent of serum-NfL. Interestingly, the extent of BBB permeability offered more robust correlations with serum-NfL measured at multiple time points, suggesting BBB disruption is an important determinant of serum biomarker dynamics after mTBI. CONCLUSIONS: These data provide novel insights to the temporal course and pathological basis of serum NfL measurements that inform its utility as a biomarker in mTBI. The ability of NfL to measure underlying pathology will be critical for future clinical and translational research.

ACE2 Rescues Sepsis-Associated Encephalopathy by Reducing Inflammation, Oxidative Stress, and Neuronal Apoptosis via the Nrf2/Sestrin2 Signaling Pathway.

Neuroinflammation and oxidative stress contribute to the progression of sepsis-associated encephalopathy (SAE). Angiotensin-converting enzyme 2 (ACE2) is considered to be a neuroprotective factor due to its anti-inflammatory and antioxidant properties. However, the role of ACE2 on myeloid cells in regulating SAE and the underlying mechanism warrants further exploration. SAE was induced in ACE2 transgenic (TG), knockout (KO), and bone marrow (BM) chimeric mice by cecal ligation and puncture (CLP). The expression levels of apoptosis-, oxidation- and neuroinflammation-associated mediators and morphological changes were monitored by quantitative real-time PCR analyses and histological examinations in the cortex of septic mice. The contents of angiotensin (Ang) II and Ang-(1-7) along with the activity of ACE2 were examined with commercial kits. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and Sestrin2 was detected by immunoblotting analysis. Our results indicated that the expression of cortical ACE2 was significantly reduced in the early phase of CLP-induced sepsis. Moreover, ACE2 overexpression in TG mice conferred neuroprotection against sepsis, as evidenced by alleviated neuronal apoptosis, oxidative stress, and proinflammatory M1-like microglial polarization, accompanied by upregulation of the Ang-(1-7), Nrf2, and Sestrin2 protein levels. Conversely, ACE2 deficiency in KO mice exacerbated SAE. The neuroprotective effects of ACE2 were further confirmed in wild-type mice transplanted with ACE2-TG and KO BM cells. Therefore, our data suggest that myeloid ACE2 exerts a protective role in the pathogenesis of SAE, potentially by activating Ang-(1-7)-Nrf2/sestrin2 signaling pathway, and highlight that upregulating ACE2 expression and activity may represent a promising approach for the treatment of SAE in patients with sepsis.

Glucocorticoid-Induced Myopathy: Typology, Pathogenesis, Diagnosis, and Treatment.

Glucocorticoid-induced myopathy is a non-inflammatory toxic myopathy typified by proximal muscle weakness, muscle atrophy, fatigue, and easy fatigability. These vague symptoms coupled with underlying disorders may mask the signs of glucocorticoid-induced myopathy, leading to an underestimation of the disease's impact. This review briefly summarizes the classification, pathogenesis, and treatment options for glucocorticoid-induced muscle wasting. Additionally, we discuss current diagnostic measures in clinical research and routine care used for diagnosing and monitoring glucocorticoid-induced myopathy, which includes gait speed tests, muscle strength tests, hematologic tests, bioelectrical impedance analysis (BIA), dual-energy X-ray absorptiometry (DXA), computed tomography (CT), magnetic resonance imaging (MRI), electromyography, quantitative muscle ultrasound, histological examination, and genetic analysis. Continuous monitoring of patients receiving glucocorticoid therapy plays an important role in enabling early detection of glucocorticoid-induced myopathy, allowing physicians to modify treatment plans before significant clinical weakness arises.

Histomorphometry of Bone after Intentionally Exposed Non-Resorbable d-PTFE Membrane or Guided Bone Regeneration for the Treatment of Post-Extractive Alveolar Bone Defects with Implant-Supported Restorations: A Pilot Randomized Controlled Trial.

Aim: The aim of the present study was to investigate quantitative histological examination of bone reconstructed with non-resorbable high-density polytetrafluoroethylene membrane (d-PTFE), left intentionally exposed in post extraction sockets grafted with anorganic bone material, and removed after four weeks, versus extraction and guided bone regeneration (GBR), performed two months later. Materials and Methods: This study was designed as a multicenter randomized controlled trial of parallel-group design. Patients were selected and consecutively treated in three centers in Italy. Patients randomly received intentionally exposed non-resorbable d-PTFE membrane (group A), or guided bone regeneration (group B), to treat post-extractive alveolar bone defects with implant-supported restorations. Outcomes were: the implant failure, any mechanical and biological complications, patient satisfaction, and qualitative and histomorphometric evaluation of the collected bone samples. Results: Eighteen patients were consecutively enrolled in the trial. Of these, six out of 18 patients were male. All the included patients were treated according to the allocated interventions, and no drop out occurred. No implant failure and no complications were experienced, and all the patients were fully satisfied with the function and aesthetic of their implant-supported restoration, without difference between groups. Morphological analysis revealed no sign of tissue reaction, such as fibrosis or necrosis. Regenerated bone was well mineralized in both groups, but it seemed more mature in group B than in group A. Three samples showed a minimal number of lymphocytes. Several blood vessels of small size occupied the medullary spaces, where the tissue resulted in more maturity, indicating the activity of the tissue in progress. The histomorphometric evaluation showed no statistically significant differences in the tissue volume fractions between the two groups of patients. Conclusions: With the limitation of the present study, buccal plate reconstruction with an intentionally exposed non-resorbable membrane is an effective and easy procedure for regenerating a resorbed buccal bone plate, reducing the need for guided bone regeneration.

Glibenclamide Directly Prevents Neuroinflammation by Targeting SUR1-TRPM4-Mediated NLRP3 Inflammasome Activation In Microglia.

Glibenclamide (GLB) reduces brain edema and improves neurological outcome in animal experiments and preliminary clinical studies. Recent studies also suggested a strong anti-inflammatory effect of GLB, via inhibiting nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation. However, it remains unknown whether the anti-inflammatory effect of GLB is independent of its role in preventing brain edema, and how GLB inhibits the NLRP3 inflammasome is not fully understood. Sprague-Dawley male rats underwent 10-min asphyxial cardiac arrest and cardiopulmonary resuscitation or sham-operation. The Trpm4 siRNA and GLB were injected to block sulfonylurea receptor 1-transient receptor potential M4 (SUR1-TRPM4) channel in rats. Western blotting, quantitative real-time polymerase chain reaction, behavioral analysis, and histological examination were used to evaluate the role of GLB in preventing NLRP3-mediated neuroinflammation through inhibiting SUR1-TRPM4, and corresponding neuroprotective effect. To further explore the underlying mechanism, BV2 cells were subjected to lipopolysaccharides, or oxygen-glucose deprivation/reperfusion. Here, in rat model of cardiac arrest with brain edema combined with neuroinflammation, GLB significantly alleviated neurocognitive deficit and neuropathological damage, via the inhibition of microglial NLRP3 inflammasome activation by blocking SUR1-TRPM4. Of note, the above effects of GLB could be achieved by knockdown of Trpm4. In vitro under circumstance of eliminating distractions from brain edema, SUR1-TRPM4 and NLRP3 inflammasome were also activated in BV2 cells subjected to lipopolysaccharides, or oxygen-glucose deprivation/reperfusion, which could be blocked by GLB or 9-phenanthrol, a TRPM4 inhibitor. Importantly, activation of SUR1-TRPM4 in BV2 cells required the P2X7 receptor-mediated Ca2+ influx, which in turn magnified the K+ efflux via the Na+ influx-driven opening of K+ channels, leading to the NLRP3 inflammasome activation. These findings suggest that GLB has a direct anti-inflammatory neuroprotective effect independent of its role in preventing brain edema, through inhibition of SUR1-TRPM4 which amplifies K+ efflux and promotes NLRP3 inflammasome activation.

Associations of Lumber Disc Degeneration With Paraspinal Muscles Myosteatosis in Discogenic Low Back Pain.

Accompanied with intervertebral disc (IVD) degeneration, increasing fat infiltration of paraspinal muscles may be related to discogenic low back pain (DLBP), but their relationship is still unclear and the classical animal models are not completely applicable. The purpose of this study was to assess the paraspinal muscle fat infiltration in patients with DLBP by quantitative MRI, and to develop a novel DLBP rat model to explore the potential relationship between DLBP paraspinal muscle fat infiltration and TNF-α levels. We measured the proton density fat fraction (PDFF) of the multifidus and erector spinae muscles of 70 DLBP patients and 36 healthy volunteers by using quantitative MRI IDEAL-IQ. In addition, we developed a DLBP experimental rat model by puncturing the L4/5 and L5/6 IVDs under the guidance of X-ray fluoroscopy. Then various behavioral experiments, MRI and pathological examination of IVDs were used to evaluate the performance of the DLBP animal model. The gait analysis, hot plate test, acetone test, grasping test and tail suspension test were used to evaluate the pain and muscle dysfunction in rats. Through quantitative MRI and histological examination, the degeneration of IVDs and fat infiltration in the muscles were observed in vivo and ex vivo. Enzyme linked immunosorbent assay detects the level of TNF-α in rat IVDs and paraspinal muscles. In the human study, compared with healthy volunteers, the PDFF of multifidus and erector muscles of DLBP patients increased significantly at L4/5 and L5/S1 levels (p<0.05). In the rat experiment, compared with control group and sham group, DLBP group had reduced gait score, shortened response time to cold and heat stimuli, prolonged bending time, and shortened struggling time. Rat lumbar MRI T2WI showed that the signal intensity of L4/5 and L5/6 IVDs were progressively decreased. Histological examination revealed that IVDs had increased collagen fibers, reduced nucleus pulposus, thickened annulus fibrosus, and distorted shape. The PDFF of multifidus muscle at L4/5 and L5/6 level in the DLBP group were more than that in other groups (p<0.05), and HE staining and oil red O staining of paraspinal muscles showed that the muscle bundle space of the DLBP group muscles increased, and the muscle tissues Increased lipid droplets. Finally, the expression of TNF-α in IVDs and paraspinal muscles in the DLBP group were significantly higher than that in the control group (p<0.05). It is reliable and feasible to establish a DLBP rat model by puncturing the lumbar IVDs under the guidance of X-ray fluoroscopy. The degeneration of lumbar IVDs with DLBP leads to the occurrence of fat infiltration of paraspinal muscles, which is related to the expression of TNF-α.

The vascular impact of dapagliflozin, liraglutide, and atorvastatin alone or in combinations in type 2 diabetic rat model.

Diabetic dyslipidemia is a significant contributor in the pathogenesis of type 2 diabetes (T2D). The study aimed at comparing the effect of dapagliflozin, liraglutide, and atorvastatin alone or their combinations on lipids and inflammatory markers and their vascular impact in T2D rats. There were 56 male albino rats included in the study and divided into two main groups. Group A (8 rats) served as normal control. Group B (48 rats) were streptozotocin-nicotinamide-induced diabetic rats. Subgroups (B-1, B-2, B-3, B-4, B-5, and B-6) received (no medications, dapagliflozin, liraglutide, atorvastatin, dapagliflozin + atorvastatin, and liraglutide + atorvastatin), respectively. Urine albumin/creatinine ratio (UACR), glycosylated hemoglobin (HBA1c), fasting serum glucose (FSG), serum low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), TGs, lipoprotein(a) Lp (a), serum thyrotropin (TSH), highly sensitive C-reactive protein (hs-CRP) and advanced glycation end products (AGEs), were assessed. Qualitative and quantitative histological examination of kidneys focused on renal corpuscles. Dapagliflozin improved the studied parameters but with statistically insignificant increase in LDL-C, Lp (a) and significant increase in UACR. Atorvastatin improved the studied parameters but with statistically insignificant increase in FSG and HbA1C. Liraglutide and the combination groups significantly improved all studied parameters. Histologically, liraglutide and atorvastatin produced therapeutic effect, while dapagliflozin depicted nephrotoxic effect. Combination groups resulted in better effects with normalization of most of renal corpuscles. There were positive correlations between LDL-C and hs-CRP, AGEs, TSH and mesangial expansion. Combination of atorvastatin with liraglutide can improve its vasculoprotective effect. Moreover, combination of atorvastatin with dapagliflozin can ameliorate its possible nephrotoxic effect.

Bone Biopsy for Histomorphometry in Chronic Kidney Disease (CKD): State-of-the-Art and New Perspectives.

The use of bone biopsy for histomorphometric analysis is a quantitative histological examination aimed at obtaining quantitative information on bone remodeling, structure and microarchitecture. The labeling with tetracycline before the procedure also allows for a dynamic analysis of the osteoblastic activity and mineralization process. In the nephrological setting, bone biopsy is indicated to confirm the diagnosis of subclinical or focal osteomalacia and to characterize the different forms of renal osteodystrophy (ROD). Even if bone biopsy is the gold standard for the diagnosis and specific classification of ROD, the use of this approach is very limited. The main reasons for this are the lack of widespread expertise in performing or interpreting bone biopsy results and the cost, invasiveness and potential pain associated with the procedure. In this regard, the sedation, in addition to local anesthesia routinely applied in Italian protocol, significantly reduces pain and ameliorates the pain perception of patients. Concerning the lack of widespread expertise, in Italy a Hub/Spokes model is proposed to standardize the analyses, optimizing the approach to CKD patients and reducing the costs of the procedure. In addition, new tools offer the possibility to evaluate the osteogenic potential or the ability to form bone under normal and pathological conditions, analyzing mesenchymal stem cells and their ability to differentiate in the osteogenic lineage. In the same way, circulating microRNAs are suggested as a tool for exploring osteogenic potential. The combination of different diagnostic approaches and the optimization of the bioptic procedure represent a concrete solution to spread the use of bone biopsy and optimize CKD patient management.

The roles of MCP-1/CCR2 mediated macrophage recruitment and polarization in bladder outlet obstruction (BOO) induced bladder remodeling

Bladder outlet obstruction (BOO) can lead to alternation of bladder structure and function, known as bladder remodeling. Macrophage is a heterogeneous cell type and implicated in immunity regulating and tissue repairment. The relationship between macrophage and BOO remains unclear. We determined the pivotal role of macrophage recruitment and polarization in bladder remodeling. Sprague-Dawley rats underwent surgical operation of a BOO for either 1, 3, 6 weeks and were compared with sham-operated rats. The BOO rats in the experimental group were orally administrated with 5 mg/kg RS-504393, a C-C chemokine receptor (CCR2) antagonist, for 6 weeks, and the rats in the control group were treated with vehicle. Bladder tissues were harvested for assays of flow cytometry, quantitative reverse transcription polymerase chain reaction, histological examinations, immunohistochemistry staining and immunofluorescence. After induction of BOO, M1 macrophages were predominantly observed at inflammatory stage while M2 macrophages were mainly found during fibrosis stage. Flow cytometry analysis revealed that the ratio of M1/M2 significantly increased at 3 weeks (P = 0.0013) when compared to the sham-operated group. Interestingly, our results showed that M2 macrophages promoted BOO-induced fibrosis through indirectly secreting TGF-β and directly transforming to collagen-producing myofibroblast. Additionally, RS-504393 treatment significantly decreased the number of M1 and M2 macrophage infiltration in bladder tissue, and bladder fibrosis was attenuated by RS-504393 treatment compared with that in the vehicle-treated rats. In summary, macrophages play a pivotal role in bladder remodeling and targeting MCP-1/CCR2 signaling pathway might be a therapeutic strategy for human bladder fibrosis.

Human Tissue Kallikrein 1 Is Downregulated in Elderly Human Prostates and Possesses Potential In Vitro Antioxidative and Antifibrotic Effects in Rodent Prostates.

Objective The aim of the present study was to investigate the protective effects and mechanisms of KLK1 on aging-related prostate alterations and search clues about the application of KLK1 to the treatment of human BPH. Methods Thirty-six rats including 26 male wild-type SD rats and 10 transgenic rats were fed to 3- or 18-month-old and divided into three groups: young WTR (yWTR) as the control (n = 16), aged WTR (aWTR) (n = 10), and aged TGR (aTGR) (n = 10). The prostates of the three groups of rats (10 rats per group) were harvested to evaluate the levels of KLK1 expression, oxidative stress, fibrosis, and involved signaling pathways, such as NO/cGMP, COX-2/PTGIS/cAMP, and TGF-β1/RhoA/ROCK1, via quantitative PCR, Western blot, histological examinations, and ELISA. Moreover, the remaining 6 yWTRs were sacrificed to obtain primary prostate fibroblast and aortic endothelial cells, and a coculture system was built with the cells for the verification of above signaling pathways in vitro. And the direct effects of bradykinin on prostate cells were detected by MTT experiment. Prostate specimens of 47 patients (age from 48 to 92 years) undergoing BPH surgery were collected after approval. Histological examinations and KLK1 IHC were preformed to analyze the relationship between KLK1 expression and age and prostate fibrosis. Results The human KLK1 gene only existed and was expressed in aTGR. The prostate of young rats expressed more KLK1 than the aged and the expression of KLK1 in prostate decreased with age in humans (r = −0.347, P = 0.018). Compared to the aWTR group, the yWTR and aTGR groups showed milder fibrosis, less oxidative stress, upregulated NO/cGMP, and COX-2/PTGIS/cAMP signaling pathways and inhibited TGF-β1/RhoA/ROCK1 signaling pathway. In the coculture system, KLK1 suppressed TGF-β1-mediated fibroblast-to-myofibroblast transdifferentiation via cleaving LMWK to produce the BK which upregulate eNOS expression and NO production in endothelial cells. BK not only slightly stimulated the proliferation ability of prostatic stromal cells but also upregulated iNOS and inhibited TGF-β1 expression in them. Conclusion KLK1 protects prostate from oxidative stress and fibrosis via amplified NO/cGMP signal in aged rats. The decrease of KLK1 expression with aging is laying the groundwork for the application of KLK1 to the treatment of human BPH. The current experimental data showed that the side effects of KLK1 on the prostate cell were not obvious.

Cranial bone flap resorption\u2014pathological features and their implications for clinical treatment

Cranioplasty following decompressive craniectomy (DC) has a primary complication when using the autologous bone: aseptic bone resorption (ABR). So far, risk factors such as age, number of fragments, and hydrocephalus have been identified but a thorough understanding of the underlying pathophysiology is still missing. The aim of this osteopathological investigation was to gain a better understanding of the underlying processes. Clinical data of patients who underwent surgical revision due to ABR was collected. Demographics, the time interval between craniectomy and cranioplasty, and endocrine serum parameters affecting bone metabolism were collected. Removed specimens underwent qualitative and quantitative histological examination. Two grafts without ABR were examined as controls. Compared to the controls, the typical layering of the cortical and cancellous bone was largely eliminated in the grafts. Histological investigations revealed the coexistence of osteolytic and osteoblastic activity within the necrosis. Bone appositions were distributed over the entire graft area. Remaining marrow spaces were predominantly fibrotic or necrotic. In areas with marrow cavity fibrosis, hardly any new bone tissue was found in the adjacent bone, while there were increased signs of osteoclastic resorption. Insufficient reintegration of the flap may be due to residual fatty bone marrow contained in the bone flap which seems to act as a barrier for osteogenesis. This may obstruct the reorganization of the bone structure, inducing aseptic bone necrosis. Following a path already taken in orthopedic surgery, thorough lavage of the implant to remove the bone marrow may be a possibility, but will need further investigation.

Stomoxys calcitrans, mechanical vector of virulent Besnoitia besnoiti from chronically infected cattle to susceptible rabbit.

Cattle besnoitiosis caused by Besnoitia besnoiti (Eucoccidiorida: Sarcocystidae) is a re‐emerging disease in Europe. Its mechanical transmission by biting flies has not been investigated since the 1960s. The aim of this study was to re‐examine the ability of Stomoxys calcitrans (Diptera: Muscidae) to transmit virulent B. besnoiti bradyzoites from chronically infected cows to susceptible rabbits. Three batches of 300 stable flies were allowed to take an interrupted bloodmeal on chronically infected cows, followed by an immediate bloodmeal on three rabbits (Group B). A control group of rabbits and a group exposed to the bites of non‐infected S. calcitrans were included in the study. Blood quantitative polymerase chain reaction (qPCR) analyses, and clinical, serological and haematological surveys were performed in the three groups over 152 days until the rabbits were killed. Quantitative PCR analyses and histological examinations were performed in 24 tissue samples per rabbit. Only one rabbit in Group B exhibited clinical signs of the acute phase of besnoitiosis (hyperthermia, weight loss, regenerative anaemia and transient positive qPCR in blood) and was seroconverted. Parasite DNA was detected in four tissue samples from this rabbit, but no cysts were observed on histological examination. These findings indicate that S. calcitrans may act as a mechanical vector of B. besnoiti more efficiently than was previously considered.

Extracellular vesicles from human umbilical cord blood ameliorate bone loss in senile osteoporotic mice

ObjectiveSenile osteoporosis is one of the most common age-related diseases worldwide. Accumulating evidences have indicated that young blood can reverse age-related impairments. Extracellular vesicles (EVs) exert therapeutic effects in a variety of diseases by delivering bioactive molecules such as microRNAs (miRNAs). The aim of the study is to evaluate the therapeutic potential of EVs from human umbilical cord blood plasma (UCB-EVs) on senile osteoporosis and to preliminarily clarify the underlying mechanism. MethodsUCB-EVs were injected into the tail vein of aged (16 months old) male C57BL/6 mice. Microcomputed tomography was performed to evaluate bone mass and microarchitecture of mice. The osteogenic and osteoclastic activities were determined by quantitative real-time PCR (qRT-PCR), histological examination and western blot analysis. In vitro, qRT-PCR assay was undertaken to explore the enrichment levels of a number of miRNAs that have positive effects in reducing bone loss. The efficacy of UCB-EVs on osteoblastic differentiation of bone marrow mesenchymal stromal cells (BMSCs) and osteoclastogenesis of RAW264.7 cells were assessed by cytochemical staining. Gene and protein expression changes were detected by qRT-PCR and western blotting respectively. Meanwhile, the roles of the selected miRNA in the regulatory effects of UCB-EVs on BMSCs and RAW264.7 cells were evaluated by using specific miRNA inhibitor. ResultsThe intravenous injection of UCB-EVs for two months attenuated bone loss in old mice, as defined by increased trabecular and cortical bone mass, enhanced osteoblast formation and reduced osteoclast formation compared to the control mice. In vitro, UCB-EVs could promote the osteogenic differentiation of BMSCs and inhibit the osteoclastogenesis of RAW264.7 cells. Moreover, it was confirmed that miR-3960 was highly enriched in UCB-EVs and miR-3960 inhibitor reversed the stimulatory effect of UCB-EVs on osteoblastic differentiation of BMSCs. ConclusionOur findings indicate that UCB-EVs ameliorate age-related bone loss by stimulating bone formation and inhibiting bone resorption, and miR-3960 mediated the osteogenic effect of UCB-EVs on BMSCs. Thus, UCB-EVs may represent a promising agent for prevention of senile osteoporosis.

Histological changes in the proximal suspensory ligament after neurectomy of the deep branch of the lateral palmar nerve of horses with induced proximal suspensory desmitis

Proximal suspensory desmitis (PSD) is a common cause of lameness in the pelvic limb, but could also affect the thoracic limb of competing and non-competing horses. Most horses diagnosed with PSD in a thoracic limb respond to rest followed by controlled exercise, but in a small percentage of affected horses, lameness persists. In one study, four horses chronically lame because of PSD in a thoracic limb became sound after neurectomy of the deep branch of the lateral palmar nerve (DBLPaN), which innervates the proximal aspect of the suspensory ligament (SL; Guasco et al., 2013). Whether neurectomy of the DBLPaN results in changes in the SL that might predispose the horse to re-injury is not known. The aim of this study was to describe the findings observed during quantitative lameness evaluation, gross and histological examination of the proximal portion of the suspensory ligament (SL) of the thoracic limbs of eight horses after neurectomy of the DBLPaN performed after inducing unilateral PSD by injecting collagenase into the proximal portion of the SL. The clinical response to neurectomy was resolution of lameness in all horses. Muscle fibers of the denervated ligaments presented atrophy and were infiltrated with fat and connective tissue, thereby reducing the strength and elasticity of the ligament.

Efficacy of the Quorum Sensing Inhibitor FS10 Alone and in Combination with Tigecycline in an Animal Model of Staphylococcal Infected Wound.

In staphylococci, quorum sensing regulates both biofilm formation and toxin production, moreover it has been demonstrated to be inhibited by RNAIII inhibiting peptide (RIP). Aim our study was to evaluate the in vitro activity and its in vivo efficacy of the combined administration of FS10, a novel RIP derivative, and tigecycline in an animal model of methicillin-resistant (MR) and methicillin-sensitive (MS) Staphylococcus aureus wound infection. Using a 1.x2 cm template, one full thickness wound was established through the panniculus carnosus on the back subcutaneous tissue of each animal. Infection was determined by inoculation of 5x107 CFU/ml of bacteria, that produced an abscess within 24 h, after this, treatment was initiated. The study included, for each strain, a control group without infection, a control infected group that did not receive any treatment and a control infected group with drug-free foam dressing, and three infected groups treated, respectively, with: FS10-soaked foam dressing (containing 20 μg FS10), daily intraperitoneal tigecycline (7 mg/Kg), FS10-soaked foam dressing (containing 20 μg FS10) and daily intraperitoneal injections of tigecycline (7 mg/Kg). The main outcome measures were quantitative culture and histological examination of tissue repair. The highest inhibition of infection was achieved in the group that received FS10-soaked and parenteral tigecycline reducing the bacterial load from 107 CFU/ml to about 103 CFU/g for MSSA and to about 104 CFU/g for MRSA. The group treated with FS10-soaked foam dressing associated with parenteral tigecycline showed, histologically, better overall healing with epithelialization and collagen scores significantly higher than those of the other groups in both strains. In conclusion, the combined use of topical FS10 with i.p. tigecycline induced positive interaction in vivo, resulting in an enhanced therapeutic benefit versus staphylococcal infections in murine wound models.

Pathophysiological changes induced by Pseudomonas aeruginosa infection are involved in MMP-12 and MMP-13 upregulation in human carcinoma epithelial cells and a pneumonia mouse model.

Pseudomonas aeruginosa infections persist in patients with cystic fibrosis (CF) and drive lung disease progression. P. aeruginosa potently activates the innate immune system mostly through the recognition of pathogen-associated molecular patterns, such as flagellin. Matrix metalloproteinases 12 and 13 (MMP-12 and MMP-13, respectively) exacerbate chronic lung infection and inflammation by promoting uncontrolled tissue rearrangements and fibrosis, yet the underlying molecular mechanisms by which this occurs remain largely unknown. In this study, we used quantitative bacteriology, histological examination, and proinflammatory cytokine levels to evaluate the effects of MMP-12 and MMP-13 on P. aeruginosa strain K-induced infection and pneumonia in H292 epithelial cells and mice, respectively. Under inflammatory stimulation, mRNA and protein expression levels of proinflammatory mediators were higher in strain K-infected mice and cells than in uninfected counterparts, in which MMP-12 and MMP-13 expression reached levels similar to those observed in epithelial cells. Moreover, we also found that the NF-κB pathway might be involved in the induction of cytokines in response to strain K infection. Taken together, these data suggest that MMP-12 and MMP-13 alter strain K infection in mice and play a role in inflammatory regulation by modulating cytokine levels.

EHMTI-0219. Quantitative histological examinations of inter- and intraindividual differences in the dural vasculature of the mouse

To examine both migraine and pulmonary hypertension (PH) we developed a hypoxic mouse model. Under hypoxic conditions (four weeks at 10% O2) mice develop typical symptoms of PH, characterized by an increased muscularization of arterial blood vessels in the lung. The vessel remodeling can be prevented by the chronic application of 5-HT2B receptor antagonists. We found that hypoxic mice also develop increased sensitivity towards the 5-HT2 receptor agonist meta-Chlorophenylpiperazine (mCPP). In contrast to the control group (normoxic mice kept at 20% O2) at low doses of 1 µg / kg body weight, mCPP triggers plasma protein extravasation (PPE) in the dura mater in hypoxic mice. Dural PPE is a quantifiable indicator of migraine-like events in animal models.

Functional and histological effects of inhaled magnesium alone or associated to fluoride: An experimental study in rats

The study was carried out to investigate the effects of inhaled Mg alone and associated with F in the treatment of bronchial hyperresponsiveness. 43 male Wistar rats were randomly divided into four groups and exposed to inhaled NaCl 0.9%, MeCh, MgSO4 and MgF2. Pulmonary changes were assessed by means of functional tests and quantitative histological examination of lungs and trachea. Results revealed that delivery of inhaled Mg associated with F led to a significant decrease of total lung resistance better than inhaled Mg alone (p<0.05). Histological examinations illustrated that inhaled Mg associated with F markedly suppressed muscular hypertrophy (p=0.034) and bronchoconstriction (p=0.006) in MeCh treated rats better than inhaled Mg alone. No histological changes were found in the trachea. This study showed that inhaled Mg associated with F attenuated the main principle of the central components of changes in MeCh provoked experimental asthma better than inhaled Mg alone, potentially providing a new therapeutic approach against asthma.