Collagenase Injection Research Articles

Treatment Trends and Cost Associated With Peyronie's Disease.

IntroductionProviders may use several treatment options for patients with Peyronie's disease; however, it is unclear whether practice patterns have evolved over recent years and if this has impacted cost.AimsTo investigate trends in the treatment of Peyronie's disease over time and the associated costs using a national, commercial insurance claims database.MethodsA retrospective cohort study was conducted using claims from the Truven MarketScan database from 2007 to 2018 for men with Peyronie's disease. Cost was estimated as either the sum of prescription oral or injectable treatment costs or as the single net cost associated with the operative procedure.Main Outcome MeasuresFrequency of use of various treatments for Peyronie's disease and associated costs were assessed as trends over the timeline by year.ResultsThe estimated annual incidence of Peyronie's disease in this population rose from 61 to 77 per 100,000 patients over the included years, and the percent annual treatment rate rose from 17.8% to 26.2%. Colchicine was the most commonly prescribed oral agent in 2007 used in 22% of treated individuals; by 2018, pentoxifylline was the most common prescribed oral agent used in 33%. In 2007, 11% of treated patients received intralesional verapamil; however, by 2018, 24% received injectable collagenase, whereas <1% received intralesional verapamil. The mean annual, per-individual cost of Peyronie's disease treatment increased from $1,531 in 2007 to $10,339 in 2018. The cost increase was greatest for injectable therapies, which rose from $811 per individual in 2007 to $16,184 in 2018, a 19-fold increase.ConclusionsDiagnosis and treatment of Peyronie's disease is increasing over time. Pentoxifylline has become the most common oral prescription, whereas injectable collagenase has become most common injection. The mean cost associated with Peyronie's disease treatment increased more than 5 times from 2007 to 2018 corresponding with Federal Drug Administration's approval of injectable collagenase.Loftus CJ, Rajanahally S, Holt SK, et al. Treatment Trends and Cost Associated With Peyronie's Disease. Sex Med 2020;8:673–678.

Mechanical Overload Followed by Consecutive Collagenase Injections: Developing a Multifactorial and Long-Lasting Animal Model of Induced Achilles Tendinopathy

Category:Basic Sciences/Biologics; Hindfoot; SportsIntroduction/Purpose:Different animal models of Achilles tendinopathy have been proposed in the literature. They usually involve the induction of tendinopathic findings by either chemical stress (most commonly with one or more injections of collagenase, mimicking intrinsic factors) or mechanical stress (by repetitive exercise-induced stress with treadmill running exercises, simulating extrinsic risk factors). To date, no study has evaluated the combination of a mechanical trigger followed by collagenase injections, replicating the logical and sequential steps involved in the development the human pathology. Our goal was to develop this novel animal model of Achilles tendinopathy and to compare histological and functional findings with animals subjected to isolated mechanical or chemical stress, as well as to controls.Methods:Sixty-four Sprague-Dawley rats were divided into four groups (n=16): isolated treadmill running protocol (15o uphill running, 20meters/minute, 1hour/day, 3 weeks duration, weeks 2-4); isolated injections of collagenase (0.1mg each, 3 injections total, weeks 5-7); treadmill protocol (weeks 2-4) followed by three consecutive collagenase injections (weeks 5-7); and controls, no running and three injections of normal saline (weeks 5-7). Five animals from each group were sacrificed at weeks 8 and 10. Six animals by group were sacrificed at week 12. Gait analysis was performed at weeks one (after acclimation), five (following running protocol), eight (following injection protocol) and twelve (just before latest sacrifice time-point). Histological findings were assessed by the Movin Tendinopathy Score (eight parameters, scored from 0-3, total score 0-24), assessing collagen arrangement, structure, and stainability, cellularity, vascularity, nuclear rounding, hyalinization and presence of glycosaminoglycans. Gait parameters included stand and swing times, stride length, duty cycle and swing length.Results:After 8 weeks, significantly increased tendinopathic scores (p<0.001) were found in animals subjected to collagenase injections (16, CI 13.1-18.9) and to running/collagenase (17.4, CI 14.4-20.3), when compared to controls (1.6, CI -1.3-4.50) and running (3, CI 0.1-5.9). Similarly, after 10 weeks significantly increased scores were found in the same groups, with slight severity regression: controls (1, CI -0.8-2.8), running (2.2, CI 0.4-4.0), collagenase (10, CI 8.2-11.8) and running/collagenase (17.6, CI 15.8- 19.4). After 12 weeks, collagenase group demonstrated reversion of the findings (3.3, CI 1.6-5.1), and wasn’t different than control (2.1, CI 0.4-3.9) and running groups (2.5, CI 0.3-4.7). However, significantly increased pathological findings were noted in the running/collagenase group (20.0, CI 18.2-21.8) consistent with chronic tendinopathic process. Gait analysis results presented in Figure1.Conclusion:When compared to other models of induced Achilles tendinopathy and to controls, the novel animal model induced by a mechanical trigger and sustained by chemical stress demonstrated progressively increased histological tendinopathic scores after 12 weeks. Findings observed after isolated mechanical or chemical stresses were temporary, not maintained at latest follow- up. Steps involved in tendinopathy development, as well as the observed histological results of the combined running/collagenase model, replicate better the findings of human chronic Achilles tendinopathy. Applications for this novel model are promising, potentially supporting a better understanding of early/late findings as well as treatment options for Achilles tendinopathy.Figure 1.

Different collagenase delivery for Dupuytren disease in public hospitals

Background: The delivery protocol of collagenase Clostridium histolyticum (collagenase) injection for Dupuytren’s disease is variable, due to limited evidence for any one approach and widespread ‘off-label’ delivery occurring in Australia. As such, this preliminary study aimed to assess whether different collagenase delivery protocols for treating Dupuytren’s disease have an impact on effectiveness and safety. It was hypothesised that different collagenase delivery would affect outcomes. Methods: This preliminary, prospective study included a consecutive cohort of adult patients with Dupuytren’s disease being treated with collagenase within two Australian public hospitals to determine whether different collagenase delivery protocols impact on effectiveness and safety. The therapeutic effect was measured objectively using the total passive extension deficit (TPED), clinical success and clinical improvement. Three patient-reported outcome measures (PROMs) were used: Unité Rhumatologique des Affections de la Main (URAM), the Southampton Dupuytren’s Scoring Scheme and the Canadian Occupational Patient-Specific Functional Scale (PSFS). Results: The delivery of collagenase was variable at both clinics. The number of patients treated with collagenase at Institute I and Institute II was 49 and 18, respectively. Clinical success was achieved in 42 per cent of the Institute I and 35 per cent of the Institute II cohort. A statistically significant reduction in all three PROMs was observed for both cohorts. No significant differences between effectiveness or safety was found when comparing the two cohorts. Conclusion: The delivery of collagenase was variable at Institutes I and II, but these differences did not appear to impact the effectiveness or safety of collagenase delivery.

The Treatment of Capsular Contracture Around Breast Implants Induced by Fractionated Irradiation: The Collagenase of the Bacterium Clostridium Histolyticum as a Novel Therapeutic Approach.

Irradiation therapy limits the utilization of silicone implants for breast reconstruction due to a significant risk for capsular contracture. The injection of the collagenase of the bacterium Clostridium histolyticum (CCH) might trivialize this risk by providing a minimal-invasive treatment option by capsular contracture degradation. However, efficacy in degrading breast implant capsules induced by fractionated irradiation remains unclear. Twenty-four rats in three groups received miniature silicone implants in a submuscular pocket. After 3D dose calculation and treatment field definition, rats of two groups underwent fractionated radiotherapy (6 × 8Gy) using a linear accelerator. A third group served as control. On day 120, one irradiated group received injections of 0.3mg/ml collagenase. Administration of plain solvent solution served as control in the two other groups. Outcome parameters included CT-imaging, histology, vessel wall analysis, immunohistochemistry, chemical collagen quantification and gene expression analysis. Fractioned irradiation leads to a significant increase in collagen deposition around silicone implants with higher capsule thickness and collagen density when comparing all groups. Additionally, significant alterations of collagen fiber deposition were evident. Vessel wall thickness was significantly increased after radiotherapy. The injection of collagenase led to a significant reduction of capsule thickness, collagen density and content. However, the collagenase application induced a significant overexpression of TGFβ1. No side effects were monitored. The CCH proved to be a safe and effective approach to degrade capsule tissue induced by fractionated irradiation in an animal model. This may pave its way for clinical application in implant-based breast reconstruction patients. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Wide-awake anesthesia in Dupuytren's contracture treated with collagenase

The injection of collagenase followed by cord manipulation is one of the most popular treatments for Dupuytren's contracture. This is traditionally performed under local anesthesia or regional nerve block potentially with sedation. Neither the treatment with collagenase, nor the wide-awake anesthesia are novel techniques for hand surgeons. Nevertheless, we report the first experience of cord manipulation using the wide-awake approach. In this prospective study, we compared the pain perception of patients who underwent wide-awake anesthesia versus traditional local anesthesia. We recorded the pain sensation on a visual analog scale (VAS) (0 to 10) during anesthetic injection, during cord manipulation and before discharge. Wide-awake anesthesia significantly reduced pain levels during anesthetic injection (p=0.003) and cord manipulation (p=0.0009). Pain levels did not differ significantly right before discharge in the two groups (p=0.54). Wide-awake anesthesia can be successfully applied to cord manipulation after collagenase injection in Dupuytren's contracture. This way, it is possible to improve the patient's subjective perspective of the procedure.

Arundic Acid (ONO-2506) Attenuates Neuroinflammation and Prevents Motor Impairment in Rats with Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a severe stroke subtype caused by the rupture of blood vessels within the brain. Increased levels of S100B protein may contribute to neuroinflammation after ICH through activation of astrocytes and resident microglia, with the consequent production of proinflammatory cytokines and reactive oxygen species (ROS). Inhibition of astrocytic synthesis of S100B by arundic acid (AA) has shown beneficial effects in experimental central nervous system disorders. In present study, we administered AA in a collagenase-induced ICH rodent model in order to evaluate its effects on neurological deficits, S100B levels, astrocytic activation, inflammatory, and oxidative parameters. Rats underwent stereotactic surgery for injection of collagenase in the left striatum and AA (2μg/μl; weight × 0.005) or vehicle in the left lateral ventricle. Neurological deficits were evaluated by the Ladder rung walking and Grip strength tests. Striatal S100B, astrogliosis, and microglial activation were assessed by immunofluorescence analysis. Striatal levels of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were measured by ELISA, and the ROS production was analyzed by dichlorofluorescein (DCF) oxidation. AA treatment prevented motor dysfunction, reduced S100B levels, astrogliosis, and microglial activation in the damaged striatum, thus decreasing the release of proinflammatory cytokines IL-1β and TNF-α, as well as ROS production. Taken together, present results suggest that AA could be a pharmacological tool to prevent the harmful effects of increased S100B, attenuating neuroinflammation and secondary brain damage after ICH.

Hemorrhage promotes chronic adverse remodeling in acute myocardial infarction: a T1 , T2 and BOLD study.

Hemorrhage is recognized as a new independent predictor of adverse outcomes following acute myocardial infarction. However, the mechanisms of its effects are less understood. The aim of our study was to probe the downstream impact of hemorrhage towards chronic remodeling, including inflammation, vasodilator function and matrix alterations in an experimental model of hemorrhage. Myocardial hemorrhage was induced in the porcine heart by intracoronary injection of collagenase. Animals (N = 18) were subjected to coronary occlusion followed by reperfusion in three groups (six/group): 8 min ischemia with hemorrhage (+HEM), 45 min infarction with no hemorrhage (I - HEM) and 45 min infarction with hemorrhage (I + HEM). MRI was performed up to 4 weeks after intervention. Cardiac function, edema (T2 , T1 ), hemorrhage (T2 *), vasodilator function (T2 BOLD), infarction and microvascular obstruction (MVO) and partition coefficient (pre- and post-contrast T1 ) were computed. Hemorrhage was induced only in the +HEM and I + HEM groups on Day 1 (low T2 * values). Infarct size was the greatest in the I + HEM group, while the +HEM group showed no observable infarct. MVO was seen only in the I + HEM group, with a 40% occurrence rate. Function was compromised and ventricular volume was enlarged only in the hemorrhage groups and not in the ischemia-alone group. In the infarct zone, edema and matrix expansion were the greatest in the I + HEM group. In the remote myocardium, T2 elevation and matrix expansion associated with a transient vasodilator dysfunction were observed in the hemorrhage groups but not in the ischemia-alone group. Our study demonstrates that the introduction of myocardial hemorrhage at reperfusion results in greater myocardial damage, upregulated inflammation, chronic adverse remodeling and remote myocardial alterations beyond the effects of the initial ischemic insult. A systematic understanding of the consequences of hemorrhage will potentially aid in the identification of novel therapeutics for high-risk patients progressing towards heart failure.

IGF-1R stimulation alters microglial polarization via TLR4/NF-κB pathway after cerebral hemorrhage in mice

Intracerebral hemorrhage (ICH) is a common neurological disease, causing severe disability and even deaths. Stem cell transplantation has been increasingly used in stroke therapy, and neural stem cells (NSCs) are a good source for stem cell transplantation. However, the regulatory mechanism of NSCs remains unclear. In this study, we examined the impact of insulin-like growth factor-1 (IGF-1) secreted by NSCs on microglial polarization following ICH in adult C57BL/6 mice. Mouse models of ICH were established by collagenase injection. ICH mice received NSC transplantation 1 h after model establishment. Firstly, the changes of microglial polarization in cerebral tissues of ICH mice were detected by immunofluorescence and ELISA. Secondly, the molecular mechanism underlying the microglial polarization was evaluated repeatedly with the application of IGF-1R siRNA and IGF-1R-mediated inhibition. We assessed the brain water content and behavioral deficits of ICH mice 12, 24, 48, and 72 h after surgery. The survival of neurons in the brain was examined using Nissl staining and TUNEL staining at 72 h. The TLR4/NF-κB pathway implicated in ICH-induced inflammation was profiled by Western blot analysis and immunohistochemistry. Finally, the changes in microglia after ICH in mice were re-examined under different doses of rhIGF-1. In summary, NSC transplantation changed microglial polarization in ICH mice. IGF-1R inhibition and knockdown reversed the therapeutic effect of NSCs, and rhIGF-1 had an anti-inflammatory effect. The results of this study suggest that IGF-1R stimulation is a potential target for stem cell-based treatment of cerebral hemorrhage and may attenuate inflammatory response in the brain after bleeding.

Rh-relaxin-2 attenuates degranulation of mast cells by inhibiting NF-\u03baB through PI3K-AKT/TNFAIP3 pathway in an experimental germinal matrix hemorrhage rat model

BackgroundMast cells play an important role in early immune reactions in the brain by degranulation and the consequent inflammatory response. Our aim of the study is to investigate the effects of rh-relaxin-2 on mast cells and the underlying mechanisms in a germinal matrix hemorrhage (GMH) rat model.MethodsOne hundred seventy-three P7 rat pups were subjected to GMH by an intraparenchymal injection of bacterial collagenase. Clodronate liposome was administered through intracerebroventricular (i.c.v.) injections 24 h prior to GMH to inhibit microglia. Rh-relaxin-2 was administered intraperitoneally at 1 h and 13 h after GMH. Small interfering RNA of RXFP1 and PI3K inhibitor LY294002 were given by i.c.v. injection. Post-GMH evaluation included neurobehavioral function, Western blot analysis, immunofluorescence, Nissl staining, and toluidine blue staining.ResultsOur results demonstrated that endogenous relaxin-2 was downregulated and that RXFP1 level peaked on the first day after GMH. Administration of rh-relaxin-2 improved neurological functions, attenuated degranulation of mast cells and neuroinflammation, and ameliorated post-hemorrhagic hydrocephalus (PHH) after GMH. These effects were associated with RXFP1 activation, increased expression of PI3K, phosphorylated AKT and TNFAIP3, and decreased levels of phosphorylated NF-κB, tryptase, chymase, IL-6, and TNF-α. However, knockdown of RXFP1 and PI3K inhibition abolished the protective effects of rh-relaxin-2.ConclusionsOur findings showed that rh-relaxin-2 attenuated degranulation of mast cells and neuroinflammation, improved neurological outcomes, and ameliorated hydrocephalus after GMH through RXFP1/PI3K-AKT/TNFAIP3/NF-κB signaling pathway.

Comparison of Cellulite Severity Scales and Imaging Methods.

Cellulite is characterized by dimpled contour alterations of the skin and is present in approximately 85% to 90% of postpubertal females. Although the pathophysiology of cellulite remains to be fully elucidated, experimental evidence indicates a multifactorial process involving the number and types of fibrous septae, microvascular dysfunction, subcutaneous inflammation, decreased dermal thickness with age, and fat deposition. Cellulite is a major cosmetic concern for many women, and a number of both noninvasive (eg, massage, cosmeceuticals, laser therapy) and minimally invasive techniques (eg, subcision, collagenase injection) have been evaluated to improve the appearance of the affected skin. However, evidence for many of these treatments is limited, largely due to the lack of a validated, convenient tool for the standardized evaluation of cellulite severity. Various imaging modalities have been employed to characterize cellulite severity and the impact of treatment, but only 2-dimensional and 3-dimensional digital photography have been adequately validated. However, in many cases, imaging findings do not correlate with subjective measures of cellulite severity. A number of cellulite rating scales have been developed; some provide only a qualitative measure, whereas others do not fully capture all clinically relevant aspects of cellulite, including the perspective of the patient. There remains an unmet need for global adoption of a validated scale that can be utilized easily by clinicians and patients in clinical and research settings. We propose features that should be included in an ideal rating scale for assessment of cellulite severity.

Efficacy and safety of dorsal penile nerve block before collagenase of clostridium histolyticum injections in peyronie's disease patients: Results from a prospective pilot study.

The intraplaque injection of collagenase from Clostridium Histolyticum (CCH) was established as an effective therapeutic alternative for selected patients with Peyronie's disease (PD). There is no consensus on the use of pre-procedure anaesthesia. The aim of this pilot study was to assess the efficacy and safety of dorsal penile block before CCH injections in reducing procedure related pain. The treatment protocol described in the IMPRESS trials was adopted. The first injection of the first cycle was given without anaesthesia, while the second after penile block. After the administration of each injection, the pain related to the procedure was evaluated with the Wong-Baker-FACES® -Pain-Rating-Scale. Thirty patients were included. Mean age 56.7 (SD: 9.61) years. Mean basal penile curvature 59.37º (SD: 18.26). The mean pain value related to the procedure measured after the first injection of the first cycle (without anaesthesia) was 5.4 (SD: 2.13), while after the second injection (with anaesthesia) was 2.5 (SD: 1.92), (p<.001). The treatment was more painful in patients with dorsal plaques (mean:6.2) than in patients with lateral plaques (mean: 4.35) (p=.01). We can conclude that penile block before CCH injection in patients with PD seems an effective and safe measure to decrease the pain related to the procedure.

The influence of skin tears following collagenase treatment of Dupuytren's disease on treatment outcomes.

This study investigated influence of skin tears on patient-reported outcomes of injection of collagenase clostridium histolyticum for Dupuytren's disease and association between extension deficit of digits before injection and skin tear after the injection. From 2016 to 2018, 391 Dupuytren's cords were treated in 184 patients in a prospective cohort study and the patients were evaluated before injection and six months after injection. Skin tears occurred in 50% of these patients. We found no significant differences in the patient-reported outcomes between patients with or without skin tears. A higher extension deficit before treatment was associated with significantly increased frequency of skin tears. We conclude that the incidence of skin tears after injection does not affect patient reported outcomes six months after collagenase injection, but the incidence of skin tears is significantly associated with the severity of pre-treatment finger extension deficits.Level of evidence: II.

Modelling the catabolic environment of the moderately degenerated disc with a caprine ex vivo loaded disc culture system.

Low-back pain affects 80 % of the world population at some point in their lives and 40 % of the cases are attributed to intervertebral disc (IVD) degeneration. Over the years, many animal models have been developed for the evaluation of prevention and treatment strategies for IVD degeneration. Ex vivo organ culture systems have also been developed to better control mechanical loading and biochemical conditions, but a reproducible ex vivo model that mimics moderate human disc degeneration is lacking. The present study described an ex vivo caprine IVD degeneration model that simulated the changes seen in the nucleus pulposus during moderate human disc degeneration. Following pre-load under diurnal, simulated physiological loading (SPL) conditions, lumbar caprine IVDs were degenerated enzymatically by injecting collagenase and chondroitinase ABC (cABC). After digestion, IVDs were subjected to SPL for 7 d. No intervention and phosphate-buffered saline injection were used as controls. Disc deformation was continuously monitored to assess disc height recovery. Histology and immunohistochemistry were performed to determine the histological grade of degeneration, matrix expression, degrading enzyme and catabolic cytokine expression. Injection of collagenase and cABC irreversibly affected the disc mechanical properties. A decrease in extracellular matrix components was found, along with a consistent increase in degradative enzymes and catabolic proteins [interleukin (IL)-1β, -8 and vascular endothelial growth factor (VEGF)]. The changes observed were commensurate with those seen in moderate human-IVD degeneration. This model should allow for controlled ex vivo testing of potential biological, cellular and biomaterial treatments of moderate human-IVD degeneration.

Anti-inflammatory action of pequi oil associated to ultrasound in tendinitis in rats: macroscopic and histological analysis

Introduction: Repeated overloads of a tendon may give rise to an inflammatory response, called tendinitis, which usually begins suddenly. Researches report that the Brazilian Northeast is common to use the pequi oil (Caryocar Coriaceum wittm) in the treatment of inflammation and other diseases. Objective: To evaluate the anti-inflammatory effect of pequi oil in tendon repair process in rats after induced tendinitis. Methods: 36 male rats were divided into groups: control, ultrasound + oil pequi and pequi oil. To induce tendinitis, it was used a intratendineous injection of collagenase in the right calcaneal tendon. The treatment consisted of daily application of ultrasound + pequi oil or just pequi oil on the tendon. Macroscopic analysis was performed with the pachymeter on 1st, 7th and 14th days. Posteriorly, the rats were sacrificed with an overdose of intravenous barbiturate, then it was dissected and removed the tendon to enable the histological analysis with Hematoxylin &amp; Eosin (HE). Results: There was a reduction in the number of inflammatory cells in the animals treated for 7 days in the groups UST + pequi oil and pequi oil compared to the control, with p &lt;0,001. At 14 days, the group UST + pequi oil showed greater number of fibroblasts than the other groups, with p&lt;0,01. Conclusion: The pequi oil accelerated the tendon repair in the animals and the treatment with US + pequi oil was the most effective because it reached good statistical significance in seven and 14 days.

Time dependent degradation of vitreous gel under enzymatic reaction: Polymeric network role in fluid properties

The viscoelastic behavior of vitreous gel is due to the presence of biopolymers in its structure. Fluid properties of the vitreous is mainly the result of interactions between the characteristics of collagen type II and Hyaluronic Acid networks. Having a better understanding of the structure of each component and their changes during aging and various diseases such as diabetes can lead to better monitoring and treatment options. We study the effects of collagenase type II on 44 samples of porcine vitreous using an in situ rheological experiment in comparison with 18 eyes in a control group injected with Phosphate Buffered Saline Solution. We analyze the behavior of each component over time in both groups. We focus on the changes of viscosity and elasticity of the collagen network within the vitreous. The results of the analysis in this study show that the changes in the fluid properties of the vitreous after collagenase injection is driven by the structural alterations of the collagen network. Creep compliance values of the collagen network are significantly higher in the first group compared to the control group one hour and twenty-four hours after the injection. In contrast, creep compliance of the HA network shows no statistically significant change one hour after the injection in both groups. The results of the reported analysis of individual components in this study support the previous findings on the alterations within the vitreous structure in its entirety.

Вплив комбінованого введення кріоконсервованих мезенхімальних стовбурових клітин та агрегатів нейральних клітин на відновлення рухової активності щурів із інтрацеребральним крововиливом

The effect of implantation of cryopreserved neural cells (NCs) within the neural cells aggregates (NCAs) together with mesenchymal stem cells (MSCs) on the recovery of rat behavioural functions and motor activity after intracerebral hemorrhage (ICH) has been studied. The ICH was simulated by stereotactic injection of collagenase into rat striatum, resulted in loss of sensitivity on the affected side, disorder in motor activity and significant asymmetry of sensory-motor deficits in animals. The cryopreserved NCs (1.5 × 106 cells) within the aggregates and cryopreserved MSCs (1.0 × 106 cells) were implanted into the lateral ventricles of rat brain. A significant recovery of tactile sensitivity, improvement in the skilled tasks performance, as well as a decrease in the asymmetry of sensory-motor deficits in the ICH rats after implantation of cryopreserved NCAs, as well as the combination of the latter with MSCs, have been experimentally established. The implantation of cryopreserved MSCs together with NCAs led to an earlier improvement in behavioural-motor functions in rats with ICH as compared to the animals received the NCAs only. Probl Cryobiol Cryomed 2020; 30(2): 169–177

MicroRNA-26a-5p alleviates neuronal apoptosis and brain injury in intracerebral hemorrhage by targeting RAN binding protein 9

Emerging evidence has unraveled the important implications of microRNAs (miRNAs/miRs) in intracerebral hemorrhage (ICH). The aim of the present study was to assess the possible regulatory role of miR-26a-5p in ICH both in vivo and in vitro. ICH model of rats was constructed using stereotactic injection of VII collagenase, and ICH condition of PC-12 cells was stimulated by hemin. Exogenous overexpression of miR-26a-5p was achieved utilizing the transfection with miR-26a-5p agomir or miR-26a-5p mimics. We detected decreased miR-26a-5p and increased RAN binding protein 9 (RANBP9) levels in perihematomal tissues of ICH rats and in PC-12 cells following ICH. While miR-26a-5p overexpression alleviated behavioral deficits and neuronal apoptosis of rats with ICH. Apoptosis-related proteins Bax, Bcl-2 and cleaved caspase-3 in perihematomal region were also measured to further confirm the inhibitory effect of miR-26a-5p on neuronal apoptosis. In ICH models in vitro, we found that miR-26a-5p overexpression significantly decreased hemin-stimulated apoptosis of PC-12 cells. Additionally, RANBP9 knockdown could suppress the apoptosis of PC-12 cells, similar to the effects of PC-12 cells transfected with miR-26a-5p mimics. With dual-luciferase reporter assay, we identified that miR-26a-5p directly targeted RANBP9. In conclusion, exogenous miR-26a-5p alleviated neuronal apoptosis and brain injury partially by targeting RANBP9, and miR-26a-5p/RANBP9 axis may be a potential target for ICH treatment.

Rodent Leukocyte Isolation and Radiolabeling for Inflammation Imaging Study.

The objective of this study was to describe to develop methods of rodent leukocyte isolation and radiolabeling for in vivo inflammation imaging. Thigh muscle inflammation was induced by injection of collagenase. Blood was collected from the jugular vein and separated by Histopaque. The collected cells were incubated in a 37°C CO2 incubator for 1~2h. After incubation, 99mTc-HMPAO and 18F-FDG were used to treat leukocytes followed by incubation for 30min. 99mTc-HMPAO and 18F-FDG labeled autologous leukocytes were injected into the tail veins of rats. The images were then acquired at various time points. Image-based lesion to normal muscle ratio was compared. After Histopaque separation, the proportion of lymphocytes was higher than that of other cell types. After CO2 incubation, the collected leukocytes were viable, while room temperature exposed leukocytes without CO2 incubation were non-viable. Granulocytes, especially, were more quickly influenced by various conditions than the mononuclear cells. Labeling efficiencies of 99mTc-HMPAO and 18F-FDG were 4.00 ± 2.06 and 1.8%, respectively. 99mTc-HMPAO- and 18F-FDG-labeled leukocytes targeted well the inflamed lesion. 99mTc-HMPAO-labeled leukocytes, but not 18F-FDG-labeled leukocytes, were found in the abdomen activity. Inflamed lesions of rats were well visualized using autologous radiolabeled leukocytes. This method might provide good information for understanding inflammatory diseases.

TREM2 activation attenuates neuroinflammation and neuronal apoptosis via PI3K/Akt pathway after intracerebral hemorrhage in mice

BackgroundNeuroinflammation is an important host defense response to secondary brain injury after intracerebral hemorrhage (ICH). Triggering receptor expressed on myeloid cells 2 (TREM2) confers strong neuroprotective effects by attenuating neuroinflammation in experimental ischemic stroke. Recent studies suggest that apolipoprotein E (apoE) is a novel, high-affinity ligand of TREM2. This study aimed to investigate the effects of TREM2 activation on neuroinflammation and neuronal apoptosis in a mouse model of ICH.MethodsAdult male CD1 mice (n = 216) were subjected to intrastriatal injection of bacterial collagenase. The TREM2 ligand, apoE-mimetic peptide COG1410 was administered intranasally at 1 h after ICH induction. To elucidate the underlying mechanism, TREM2 small interfering RNA (siRNA) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 were administered intracerebroventricularly prior to COG1410 treatment. Neurobehavioral tests, brain water content, immunofluorescence, western blotting, and Fluoro-Jade C- and terminal deoxynucleotidyl transferase dUTP nick end labeling staining were performed.ResultsEndogenous TREM2 expression was increased and peaked at 24 h after ICH. TREM2 was expressed on microglia, astrocytes, and neurons. COG1410 improved both short-term and long-term neurological functions, reduced brain edema, inhibited microglia/macrophage activation and neutrophil infiltration, and suppressed neuronal apoptotic cell death in perihematomal areas after ICH. Knockdown of endogenous TREM2 by TREM2 siRNA aggravated neurological deficits and decreased the expression of TREM2 in naïve and ICH mice. COG1410 was associated with upregulation of TREM2, PI3K, phosphorylated-Akt, and Bcl-2 and downregulation of TNF-α, IL-1β, and Bax after ICH. The neuroprotective effects of COG1410 were abolished by both TREM2 siRNA and PI3K inhibitor LY294002.ConclusionsOur finding demonstrated that TREM2 activation improved neurological functions and attenuated neuroinflammation and neuronal apoptosis after ICH, which was, at least in part, mediated by activation of PI3K/Akt signaling pathway. Therefore, activation of TREM2 may be a potential therapeutic strategy for the management of ICH patients.

Acute symptomatic seizures following intracerebral hemorrhage in the rat collagenase model

Background and purposeIntracerebral hemorrhage (ICH) is a known risk factor for the development of seizures, but little is known about the pathophysiology of seizures in the acute phase post-ICH and their influence on functional outcome. With the use of an animal model, the underlying pathophysiology could be further unraveled. The aim of our study was to optimize the rat collagenase stroke model for the detection of acute symptomatic seizures using video-EEG monitoring. MethodsMale Sprague-Dawley rats were implanted with scalp electrodes and a craniotomy was made for later injection of collagenase. After one week of baseline video-EEG recording, rats were injected with 0.6 U collagenase in 0.7 μL saline in left striatum, in close proximity of the piriform cortex, and immediately reconnected to the video-EEG setup for 7 days. Occurrence of clinical and electrographic seizures was assessed and functional deficits were evaluated on several time points using the cylinder test, Neurological Deficit Scale (NDS) and forelimb placing test. At day 7 post-ICH, animals were euthanized. The volume and cortical involvement of the hemorrhage were assessed by histological examination of the brain tissue, using Cresyl violet stain. ResultsCollagenase injection induced ICH in all animals with a mean volume of 27 mm³ (SEM 7 mm³, range 4–92 mm³). Functional deficits were present in all animals injected with collagenase (pre-ICH vs post-ICH, p < 0.001). Epileptic seizures occurred in 5/11 animals and started between 1 and 61 h after ICH induction. Behavioral changes were observed in 13/15 seizures. ConclusionsInjecting rats with 0.6 U of collagenase is a useful model to study the occurrence of acute symptomatic seizures post-ICH as it results in ICH in all animals without mortality, 45% incidence of ICH-induced acute symptomatic seizures and measurable functional deficits.