Ischemic stroke presents a major global economic and public health burden. Although recent advances in available endovascular therapies show improved functional outcome, a good number of stroke patients are either ineligible or do not have access to these treatments. Also, robust collateral flow during acute ischemic stroke independently predicts the success of endovascular therapies and the outcome of stroke. Hence, adjunctive therapies for cerebral blood flow (CBF) enhancement are urgently needed. A very clear overview of the pial collaterals and the role of genetics are presented in this review. We review available evidence and advancement for potential therapies aimed at improving CBF during acute ischemic stroke. We identified heme-free soluble guanylate cyclase activators; Sanguinate, remote ischemic perconditioning; Fasudil, S1P agonists; and stimulation of the sphenopalatine ganglion as promising potential CBF-enhancing therapeutics requiring further investigation. Additionally, we outline and discuss the critical steps required to advance research strategies for clinically translatable CBF-enhancing agents in the context of acute ischemic stroke models. Stroke is the global leading cause of debilitating long-term disability and accounts for approximately 6 million deaths per annum , 2. The Despite these notable developments in the clinical management of acute ischemic cases, there remains a considerable need for alternative and adjunctive treatments. While most clinical centers have upgraded the therapeutic window for MT to 24 h from symptom onset, only a minor proportion of ischemic stroke patients are eligible for this treatment . Half ofPrevious clinical trials  for otheHere, we highlight the importance of collateral flow and the role of genetics in acute ischemic stroke. We review the potential pharmacological and adjunctive approaches reported to enhance CBF in experimental and clinical acute ischemic stroke settings. Next, we discuss the evidence and limitations of these “would be” therapies in the context of their relevance to clinical stroke comorbidities and functional recovery.The cerebral collateral circulation, an anastomosis between the tributaries of major cerebral arteries,  helps maFurther, effective leptomeningeal collateral flow may influence the rate of reperfusion and recanalization following endovascular treatments , 23, 27.Given the existence of considerable morphological variation in the quantity, diameter, and functional recruitment of collateral vessels in humans , some paPreclinical evidence from inbred mouse strains have shown that inter-individual variability of collateral extent in the brain depends chiefly on genetic makeup –38. Kao Furthermore, Cristofaro et al.  demonstrTaken together, genetic factors determine the extent and quantity of collateral vessels available for recruitment following stroke. However, genetic disposition alone cannot account for the effective recruitment of collateral vessels following cerebral ischemia. Hence, increased efforts in investigating promising adjunctive CBF-enhancing therapies are urgently needed.In addition to the time taken to arrive at the hospital, it takes over 30 min to administer medical treatments for ischemic stroke due to radiological determination of rt-PA and mechanical treatment eligibility . Given t1. Head positionHead position lowered with reference to elevated body following stroke has been associated with increased blood flow to the ischemic region . The preA follow-up report  from the2. NeuroprotectantsSphingosine-1-phosphate (S1P) belongs to the sphingolipid class of cell membrane-derived lipids and is essential for many cellular processes such as cell growth, survival, motility, angiogenesis, leucocyte migration, and maintaining BBB integrity , 59. S1PRecently, using mouse descending aorta, in vivo and in vitro, Jung et al.  showed t3. Stimulation of the sphenopalatine ganglionIntracranial blood flow increases by approximately 40% following the stimulation of the sphenopalatine ganglion (SPG), a structure responsible for the vasodilation of intracranial blood vessels via parasympathetic innervation from the superior salivatory nucleus –71.The SPG, situated in the pterygopalatine fossa of the skull, supplies parasympathetic innervation to anterior cerebral and meningeal blood vessels , 71. PreFollowing a 2 h transient  middle cerebral artery occlusion (MCAO) in Wistar rats, SPG stimulation administered 18 h after MCAO induction significantly reduced brain lesion and improved functional outcome on day 8 but not 28 . This suSimilarly, SPG stimulation administered 15 min or 24 h after photothrombotic model of permanent MCAO (pMCAO) induction in adult rats reduced infarct volume and BBB dysfunction . SPG stiTo demonstrate the importance of SPG to ischemic stroke outcome, several studies –77 reporHence, SPG stimulation bears no safety concerns but holds significant promises for improving stroke outcomes when administered prior to, and/ or following available reperfusion therapies. Further investigation at the preclinical level should consider aging and comorbidities while clinical trials should expand the number of recruited patients through well-advocated multiple-center studies which will cover areas with multi-ethnic representations. Also, it is pertinent to explore the possible interaction between thrombolytic agents and SPG stimulation, and to better understand the contributions of the different neurotransmitters released by the SPG .4. Remote ischemic perconditioningRemote ischemic conditioning was first described in 1986 in the context of myocardial infarction . HoweverRecently, pre-clinical reports indicate that RIPerC administered by three cycles of bilateral femoral artery occlusion commencing within 1 h of permanent distal middle cerebral occlusion (dMCAO) in normotensive, young adult- as well Two prior randomized trials which investigated the potential of RIPerC, as an adjunctive therapy to intravenous rt-PA for acute ischemic stroke, found that treatment administration was feasible in over 240 patients en route to the clinic  and in 1Certainly, the RIPerC treatment releases humoral factors which mediate the dilation of collateral flow, but also must have various other mechanisms for reducing infarct volume. Previous findings show increased circulating nitrite  during R5. Vasoactive agentsVarious vasodilating agents have been suggested to increase CBF following stroke. The prominent examples are nitric oxide (NO) donors, inhaled NO, soluble guanylate cyclase (sGC) activators and stimulators, rho-kinase inhibitors as well as hemoglobin-based oxygen carriers. These target vasoactive agents will be discussed under two broad sections, NO-dependent and NO-independent vasodilators, as they relate to CBF increases in acute ischemic stroke.6. NO-dependent vasodilatorsNO is a potential therapeutic agent for ischemic stroke and many experimental studies showed that exogenous NO, either via the administration of NO derivative , 97 or bNitric oxide’s role in vasodilation was first recognized in the 1970s and was then referred to as endothelium-derived relaxing factor , 101. Ab7. NO-independent vasodilatorsNewer compounds are in place to circumvent NO-signaling for vasodilation and platelet inhibition by specifically targeting the oxidized and heme-free forms of sGC, which are the abundant subtypes in chronic hypertension . IncreasTherefore, enhancing collateral recruitment and flow is critical for maximizing the amount of salvageable brain tissue and improving functional recovery. Here, we consider potential treatment strategies that may enhance the recruitment of collateral flow in the setting of acute ischemic stroke.These fundamental research reports clarified the role and capabilities of NO during ischemic stroke and led to seminal reports on pre-clinically administered NO via direct inhalation or through NO donor agents to ameliorate the effect of brain ischemia –99.Preclinical publications demonstrate the efficacy and potential benefits of glyceryl trinitrate (GTN) as an adjunctive therapy for ischemic stroke , 97.A recent study investigated the effect of sub-dermal administration of GTN (0.2 mg/h and 0.69–50 µg/h), 2 h after the induction of permanent MCAO in sheep and mice, respectively . They reNevertheless, all the phase II clinical trials in the UK and elsewhere did not attain the pre-determined desirable outcomes –108. AcrTerpolilli et al.  showed tSpecifically, hypertension is associated with decreased expression of sGC and an attenuated vascular response following the administration of NO-donors , 119–125Moreover, oxidative stress is particularly increased in the brain of SHRSPs , 127. A In light of the aforementioned, it is conceivable that NO signaling is compromised due to exaggerated oxidative stress following focal cerebral ischemia in chronic hypertension. Hence, increasing the bioavailability of NO, by inhalation or the administration of NO donor, may not translate to improvements in the recruitment of collateral blood vessels following stroke in these models—and by extension in patients with a history of chronic hypertension.2+-sGC [3+- and heme-free sGC, rather than sGC-stimulating compounds that target the reduced (Fe2+) form of sGC, are ideal for enhancing collateral flow following focal ischemic stroke in comorbid models [Stroke comorbidities such as hypertension and diabetes elevate oxidative stress, which leads to the oxidation of Fe2+-sGC . Therefod models . These Nd models , 133. Ind models  showed t2+-sGC, which are less abundant in essential hypertension. Nevertheless, by the selective release of O2 to ischemic brain tissue with low arterial oxygen tension and saturation, Sanguinate is potentially able to maintain the penumbra during occlusion as well as preserve neuronal integrity following reperfusion. In summary, it is clear that the potent vasodilatory capacity of Sanguinate is independent of NO-signaling and the exact mechanism by which Sanguinate dilates cerebral vessels following focal ischemic stroke in hypertensive and normotensive models is subject to further investigations.Sanguinate, a non-NO-dependent selective vasodilator was reported by Cipolla and colleagues  to signiIt is important to determine the evolution of penumbra as well as the dynamic recruitment of collateral flow following the administration of these NO-independent heme-independent sGC activators as well as Sanguinate in models of stroke comorbidities.Rho-associated protein kinase (ROCK) consists of two cellular subtypes, ROCK1 and ROCK2, which are both expressed in the brain and regulate basal tone of peripheral and cerebral blood vessels  by inhibFasudil, a non-specific inhibitor of ROCK1 and 2, inhibits phosphorylation of MLCK, increases eNOS expression, and causes vasodilatation of cerebral blood vessels . In vivoMoreover, Lee and colleagues  carried To date, the studies investigating the effect of ROCK inhibition on CBF usually administer treatment prior to stroke induction. Since stroke can only be clinically managed following symptom onset, potential stroke therapies are only translatable if administered following ischemic stroke. Given the promising effect of Fasudil on CBF enhancement, more studies are urgently required to investigate the CBF impact of Fasudil following MCAO induction. Indeed, one study reported that low-dose Fasudil did not increase CBF during MCAO in hypertensive rats . While tOverall, the viability of cerebral collateral flow following ischemic stroke indicates the feasibility of enhancing collateral flow as a therapeutic strategy to preserve penumbral tissue before and/ or following clinical intervention with thrombolysis and thrombectomy. While CBF enhancement is essential in acute stroke care, increased oxygen delivery to the penumbra is equally important. The pre-hospital administration of specific brain oxygen-increasing therapeutics such as normobaric hyperoxia  in combiAn ideal pre-hospital therapeutic strategy to enhance collateral circulation should satisfy the following ideal conditions: ease of administration, pharmacological safety, and rapid beneficial effect , 153. NeAdvanced age is associated with reduced collateral quantity, lumina diameter, and functional recruitment . AdditioInvestigating CBF changes often require the administration of general anesthesia to preclinical models. However, some anesthesia can lower blood pressure and induce cerebral dilatation  as Finally, some comorbidities such as chronic essential hypertension and acute post-stroke hyperglycemia induce a rapid evolution of the penumbra and expansion of the infarct volume in rodent models of ischemic stroke –172. So,We have enumerated several promising avenues to improve cerebral blood flow in acute ischemic stroke. Most stroke patients often present with more than one comorbidity , yet more studies continue to use young and otherwise healthy preclinical models to investigate potential strategies to improve CBF. While we realize that young animals can serve a vital role for the screening of potential therapies, we reason that the improvement of translational impacts of experimental ischemic stroke efforts will be best served when advanced age with chronic hypertension and other acute/ chronic comorbidities of stroke are incorporated in the testing of potential adjunctive therapies to enhance CBF Fig. .Fig. 2Im


Inflammation is the host reaction against various external and internal stimuli such as pathogen infection, tissue injury and/or dysregulated immune response. Numerous studies mostly from mammals have identified several crucial sensors and associated downstream signaling pathways that evoke inflammatory responses. Nonetheless, a full understanding of the underlying molecular mechanisms is still far from understood, because of the complex cross-talk between the innate/adaptive/stress immune responses and the implication of other biological processes such as metabolism. Insects solely rely on innate immunity and have contributed numerous important findings in the field, having a high potential to uncover new aspects of inflammatory reactions.This Research Topic explores recent advances regarding inflammatory-like reactions in insects. As Research Topic Editors, it was our great pleasure to curate and review a number of interesting manuscripts in which cover a wide range of innate immune and inflammatory-like responses across different infection models and versatile methodologies. In this Editorial, we would like to summarize the main findings and perspectives from the accepted articles in chronological order.Caravello et al. systematically investigated the contribution of a wide range of Drosophila immune defenses following infection with the obligate intracellular parasite Tubulinosema ratisbonensis (Microsporidia group). Results from the functional genetic approach indicated that phagocytosis acts as a resistant defense mechanism to control the parasite infection. In contrast, surprisingly, many of the classical antimicrobial defenses such as the systemic immune response mediated by the Toll and IMD pathways, extracellular reactive oxygen species, thioester proteins, xenophagy and intracellular antiviral response pathways were not involved in the resistance. Several genes such as PGRP-LE and Prophenoloxidase1 (PPO1) were found to promote parasite replication, while and PPO2 and Eiger were involved in resilience/disease tolerance. In conclusion, this study provided a large unexpected view of classical innate immune defenses to infection with T. ratisbonensis.in vivo Zika virus (ZIKV) infection model in Drosophila, Tafesh-Edwards et al. examined the host metabolic changes to the ZIKV infection. They found that flies deficient for Dicer-2, a central component of the antiviral RNAi pathway exhibit dysregulated glucose and glycogen metabolism and reduced insulin signaling pathway. Accordingly, insulin receptor substrate chico mutants displayed higher susceptibility and elevated ZIKV copy numbers, suggesting Dicer-2 is involved in insulin-mediated antiviral response to the ZIKV infection. Interestingly, the data also showed that these phenotypes are more severe in females than males, proposing that sex-dependent metabolic changes should be considered in the rates of infection and susceptibility to the progression of ZIKV-mediated disease.Using Deng et al. performed an integrated time-course transcriptomics and lipidomics analysis and elegantly demonstrated that ergosterol, the main sterol in the plasma membrane in flies, promotes host defense against infection with the mild-pathogen Gram-negative bacterium P. carotovorum . In particular, artificial increase of ergosterol by oral feeding or genetic depletion of Acsl, a long-chain fatty acyl-CoA synthase, significantly enhanced host survival rate to bacterial infection. Thus, this study revealed a critical role of lipid metabolism adaptation to bacterial infection and shed a light on a previously unidentified Acsl-ergosterol metabolism in innate immune response.Indeed, it is still poorly characterized how animals adjust their lipid metabolism in response to pathogenic infections. Hua et al. provided a more mechanistic insight how the IMD pathway is negatively regulated by the deubiquitinase (Dub) dTrbd which targets K63-linked ubiquitination of dTak1. Using Co-IP and GST pull-down assays, the authors showed that the N-terminal NZF domain of dTrbd is required for a physical interaction with dTak1. Ubiquitination/deubiquitination assays and phase separation analyses demonstrated that the Linker region (LR) of dTrbd modulates its condensation and contributes to regulate the Dub enzymatic activity. Interestingly, authors also observed that dTrbd/dTak1 interaction and dTrbd condensation and its Dub enzymatic activity are enhanced upon bacterial infection, suggesting dual mechanisms of dTrbd to regulate the IMD pathway activation.Vaibhvi et al. ambitiously tested the hypothesis that two innate immune cell types, namely the fat body and hemocytes, show similar immune responses to a systemic bacterial infection. In spite of a technical difficulty to isolate adult hemocytes, authors succeeded in setting up the experimental conditions and performed transcriptomic analysis on both cell types. This comprehensive analysis surprisingly found that only 10% of genes is shared between hemocytes and fat body cells, and that genes are mainly categorized into classical immune effectors or antimicrobial peptide (AMP) genes. Fat body-specific differentially regulated genes were found to involve in translation and protein export along with GO terms of oxidative reduction and phosphorylation, which is indicative of energy-consuming and metabolically active status of the fat body. In contrast, hemocytes showed a signature related to phagocytosis. In conclusion, two major immune competent cell types showed a different response to infection, aligning well with the specific tasks of humoral and cellular immunity, respectively.Finally, The findings of these studies significantly change the concept of classical innate immune responses and extend to a better understanding of insect inflammatory-like reactions. We appreciate all the excellent contributions to our Research Topic, which we hope will stimulate novel and exciting studies in the field of insect immunology.AG wrote the editorial with SJ, SC and TK. All authors approved the submitted version.


Because of its poor intrinsic repair capacity, articular cartilage seldom heals when damaged.Regenerative treatment is expected for the treatment of articular cartilage damage, and allogeneic chondrocytes or cartilage have an advantage over autologous chondrocytes, which are limited in number. However, the presence or absence of an immune response has not been analyzed and remains controversial. Allogeneic-induced pluripotent stem cell (iPSC)–derived cartilage, a new resource for cartilage regeneration, reportedly survived and integrated with native cartilage after transplantation into chondral defects in knee joints without immune rejection in a recent primate model. Here, we review and discuss the immunogenicity of chondrocytes and the efficacy of allogeneic cartilage transplantation, including iPSC-derived cartilage.Allogeneic iPSC-derived cartilage transplantation, a new therapeutic option, could be a good indication for chondral defects, and the development of translational medical technology for articular cartilage damage is expected. Articular cartilage consists of chondrocytes embedded in an abundant extracellular matrix (ECM), which is composed of type II, IX, and XI collagen molecules and proteoglycans. The ECM enables the mechanical functions of the cartilage. Cartilage has a limited regenerative capacity, and its damage tends to result in degenerative conditions, impairing joint function. Microfractures and autologous osteochondral transplantation have been used for relatively small defects  have been widely studied in allogeneic cartilage tissue transplantation. PJAC is composed of minced live cartilage allografts from juvenile donors containing chondrocytes within their native extracellular matrix. Juvenile cartilage is considered less immunogenic, and minced cartilage allows chondrocytes to diffuse out of the ECM to form new hyaline-like cartilage , 26, 28.Particulated juvenile allograft cartilage products  have been available since 2007. By 2015, more than 8700 patients had been treated with DeNovo NT . This prIn a study on iPSC-derived chondrocytes, a new resource for cartilage regeneration, iPSC-derived chondrocytes showed limited HLA expression and did not induce lymphocyte proliferation in a mixed lymphocyte assay when co-cultured with allogeneic peripheral blood mononuclear cells . TreatmeAllogeneic transplantation of iPSC-derived cartilage was performed in a primate model by mismatching the MHC, which is structurally similar to HLA, to verify the immune response in vivo. Cartilages generated from cynomolgus monkey iPS cells (cyiPSCs) were transplanted into chondral or osteochondral defects in the femoral trochlea of MHC-mismatched monkeys without the use of immunosuppressive drugs. Four weeks after allogeneic transplantation, although the graft remained intact, an accumulation of CD3-positive T cells was observed around the graft in osteochondral defects. In contrast, in chondral defects, the graft is engrafted without lymphocyte accumulation , 46. It Allogeneic transplantation of cyiPSC-derived cartilage for chondral defects showed that the cyiPSC-derived cartilage was engrafted and contributed directly to hyaline cartilage-rich repair 4 months after transplantation .FGF18 in the perichondrium-like membrane of human iPSC-derived cartilage. The addition of FGF18 promoted the integration of cartilages, whereas the addition of FGFR inhibitors inhibited it. These suggested that FGF18 secreted from the perichondrium-like membrane is involved in the integration of the human iPSC-derived cartilage [Integration of the graft and host cartilage is essential for successful tissue replacement as it provides stable biological fixation and load distribution as well as adequate mechanotransduction necessary to maintain homeostasis . Howeverartilage .Prg4 expression in mouse chondrocytes, suggesting that Sik3 negatively regulates Prg4 expression [Articular cartilage is a layered tissue that expresses proteoglycan 4 (PRG4), which functions as a lubricant in the superficial layer –57. Postpression .Allogeneic cyiPSC-derived cartilage transplantation for osteochondral defects showed that lymphocytes clustered around the graft 4 weeks postoperatively , 46; howB2M−/− cyiPSC-derived cartilage knocking out β2 microglobulin, which does not express MHC class I, was transplanted into osteochondral defects and showed immune response by NK cells [The results of allogeneic iPSC-derived cartilage transplantation in a primate model with an immune system similar to that of humans suggested that chondral defects are a better indication for allogeneic iPSC-derived cartilage transplantation than osteochondral defects. Regarding osteochondral defects, an immune response has been observed in cases of MHC mismatches, which remains a long-term concern. One solution to prevent immune reactions is to minimize rejection by matching the HLA types of the donor and host cells. iPSC lines are established from donors whose major HLA types are homozygotes , 60. TheNK cells . This reNK cells . Recent NK cells –67. CurrTransplantation of autologous iPSC-derived grafts is desirable in terms of avoiding immune reactions and minimizing the risk of spreading communicable viral infections; however, cost and manufacturing lead time is not practicable for commercialization. When the world’s first autologous transplant of iPSC-derived retinal pigment epithelial (RPE) cells was performed, the patient had to wait for more than 10 months from harvesting skin tissue to RPE sheet transplantation, which cost nearly 100 million yen , 68. On Chondrocyte and cartilage transplantation are treatment options for cartilage defects. Although chondrocytes have been shown to be hypoimmunogenic in vitro, allogeneic chondrocyte transplantation is controversial due to reports of immunoreactivity in vivo. In contrast, allogeneic cartilage transplantation, as performed in clinical practice, tends to show promising results in the short term; however, the evidence is insufficient. There are two types of cartilage defects, osteochondral and chondral defects, which may differ in their immune responses during allogeneic transplantation. The immune response to chondrocytes can be triggered by contact with the bone marrow. Although the effect of immune reactions on clinical outcomes in osteochondral defects has not yet been determined, immune reactions should be controlled to ensure good repair. Allogeneic iPSC-derived cartilage transplantation, a new therapeutic option, could be a good indication for chondral defects without an immune response. HLA type matching or iPSC lines in which HLA genes are edited can provide a solution to suppress the immune response in osteochondral defects Fig. .Fig. 2So


Current literature reports varied significance of ulnar styloid fractures (USF) associated with distal radius fractures. Our study assesses the role of ulnar styloid fractures and fragment size in surgically managed distal radius fractures.We reviewed patients who underwent surgical fixation of distal radius fractures between January 2004 to June 2006. Patients were divided into those with (Group 1) and without (Group 0) USFs. Post-operative radiographic parameters, clinical outcomes and overall wrist function were analysed. Outcomes included ulnar-sided wrist pain, extensor carpi ulnaris (ECU) tendinitis, triangular fibrocartilage complex (TFCC) grind test, distal radioulnar joint (DRUJ) instability and pain. Overall wrist function was assessed with range of motion and Disabilities of the Arm, Shoulder and Hand (DASH) score.Our study cohort included 31 males and 23 females, and 38.9% of these patients had concomitant USFs. There was no difference in terms of demographic data and fracture configuration between groups. Radiographic parameters were similar, except for palmar tilt, which was significantly higher in Group 1 . At 24 months, there were no differences in clinical outcomes and overall wrist function. A sub-group analysis showed that mean USF fragment size was larger in patients with a positive TFCC grind test .The presence of USFs in surgically managed distal radius fractures does not compromise clinical and functional outcome. Similarly, the size of USFs does not impact clinical and functional outcome but is associated with the presence of a positive TFCC grind test. However, the consensus on concomitant ulnar styloid fixation remains unclear.Age, amongst selected radiological parameters, has been well established as an important indicator for surgery when managing patients with a distal radius fracture, as recent studies have shown no improvement in outcomes after distal radius fixation in geriatric patients3. However, despite reporting that ulnar styloid fractures result in poorer outcomes of distal radius fractures, studies did not show that fixation of ulnar styloid fractures yielded superior outcomes4. Hence, a large proportion of associated ulnar styloid fractures have been conservatively managed as some studies showed that they had comparable outcome scores, range of motion, grip strength and ulnar-sided symptoms when compared to distal radius fractures without ulnar styloid involvement7.There is a high prevalence of an ulnar styloid fracture seen in distal radius fractures, with studies reporting occurrences as common as more than 50% of casesCurrent literature seems to report varied significance of ulnar styloid fractures associated with distal radius fractures, and there is a paucity of studies investigating the effects of ulnar styloid fracture fragment size in patients with surgically managed distal radius fractures. Our study aims to assess the importance of ulnar styloid fractures and ulnar styloid fragment size in surgically managed distal radius fractures. We hypothesise that the fragment size of an ulnar styloid fracture, rather than its presence, has implications in the outcomes of patients with surgically managed distal radius fractures.8. These patients were then divided into those with (Group 1) and without (Group 0) ulnar styloid fractures. Treatment of distal radius fractures in our institution were left to the discretion of individual surgeons, and all surgeries in this study were performed by fully trained orthopaedic surgeons.This was a prospective review of a retrospective cohort of patients who underwent surgical fixation of distal radius fractures in our institution between January 2004 to June 2006. Inclusion criteria were patients aged 18 years or above requiring open reduction internal fixation of a distal radius fracture. Open fractures, poly-trauma with other concomitant upper extremity injuries, fractures involving the ulnar shaft, or surgically managed ulnar styloid fractures were excluded from this study. Institutional review board approval was obtained with the need for informed consent waived. We reviewed the clinical records of 54 patients and collected demographic data including age and gender. The AO classification of each fracture, based on the distal radius classification, was also documentedStandard anteroposterior and lateral views on plain radiographs of the injured wrist before surgery, post-surgery, and at six months after surgical intervention were reviewed. The size of the ulnar styloid fragment was measured using the pre-operative radiograph, measuring the distance from the tip of the fragment to the base of the fragment . We meas9. Overall wrist function was assessed by an independent assessor in terms of range of motion and the Disabilities of the Arm, Shoulder and Hand (DASH) score. DASH questionnaires evaluate patient-reported global disabilities of the upper extremity via a 30-item questionnaire, where patients rate difficulties in activities of daily living on a 5-point scale. The test score ranges from 0 to 100, with a higher score indicating a higher level of disability. The DASH questionnaire is not specific to the wrist joint but is the most commonly used outcome measure to assess the wrist joint13.We compared outcomes between both groups at 24 months post-surgery, including ulnar-sided wrist pain, extensor carpi ulnaris (ECU) tendinitis, and the TFCC grind test for clinical evidence of possible TFCC injury. Presence of ulnar-sided wrist pain was recorded when foveal pain was elicited on palpation. Pain over DRUJ was elicited on palpation of involved DRUJ during clinical examination. DRUJ instability was assessed in terms of the “piano key” sign. ECU tendinitis was assessed clinically by localised swelling with tenderness from the ulnar styloid to the base of the 5th metacarpal, in the presence of pain reproduced on resisted active extension and ulnar deviation of the wrist. Any weakness denoting possible rupture was also noted. A positive TFCC grind test is achieved when wrist in maximum ulnar deviation, and axial stress produces ulnar wrist pain during passive supination-pronationAll patients were independently assessed in an outpatient clinic by two trained Orthopaedic residents. Pearson chi-squared test was used to compare categorical data, while the student’s t-test was used to analyse continuous variables. Statistical analyses were performed with SPSS v23.0  software and statistical significance was defined as p<0.05.Our study cohort included 31 males and 23 females with a mean age of 50.9 years . 21 out of the 54 (38.9%) patients had concomitant ulnar styloid fractures. The follow-up rate was 100% at 2 years. When comparing both groups, there was no significant difference in terms of age , gender distribution  and fracture configuration based on the AO classification (p=0.389) .On the last available radiographs at six months post-surgery, all distal radius fractures had united. There was no significant difference in radial inclination , positive ulnar variance , and the presence of dorsal comminution  between the two groups. However, palmar tilt was significantly higher in Group 1 . Both groups had minimal radial shortening and intra-articular step-off . Most ulAt 24 months, the presence of ulnar styloid fractures had no impact on ulnar-sided wrist pain (p=0.331), TFCC grind test (p=0.917), distal radioulnar joint instability (p=0.957), and pain over the distal radioulnar joint (p=0.225). None of the patients had clinical evidence of ECU rupture, but there was a tendency towards ECU tendinitis in Group 1 , but this did not achieve statistical significance. There was no significant difference in range of motion and DASH scores  .A sub-group analysis was also performed on subjects in Group 1, with the list of USF sizes of each patient listed in The clinical significance of a concomitant ulnar styloid fracture in distal radius fractures has not been clearly determined in current literature and there exists conflicting recommendations regarding management options. The main finding in our study was that the presence of an ulnar styloid fracture did not have any impact on ulnar-sided symptoms in patients who underwent surgical fixation of distal radius fractures. However, we found that conservatively managed ulnar styloid fractures with a significant fragment size is likely to result in a positive TFCC grind test result after distal radius fixation.14. Souer et al found that ulnar styloid base fractures had no significant effect on radiographic evaluation and overall outcome, other than an association with poorer grip strength and less flexion post-operatively. The difference in methodology could account for this difference as they categorised patients with ulnar styloid tip fractures in the group with “no fractures”, which could be a potential confounder15. On the contrary, a retrospective study by Ayalon et al found that patients with ulnar styloid fractures had more pain and poorer DASH scores in 184 surgically managed distal radius fractures16. However, this did not meet the minimal clinically important difference (MCID) of 10 for DASH score17. In a smaller study by Amorosa et al found that 41 out of 58 patients with concomitant ulnar styloid fractures had worse DASH scores which met the MCID of 10, but the study cohort was heterogeneous in nature with mixed treatment types rendered for distal radius fractures18. Further, even though selected studies suggest poorer outcomes with concomitant ulnar styloid fractures, there are no studies to date which has shown that fixation of ulnar styloid fractures has resulted in improved outcomes. To our knowledge, this is the only study that includes only surgically managed distal radius fracture, finding that there is no association between concomitant ulnar styloid fractures and radiographic parameters, post-operative outcome scores, as well as ulnar-sided symptoms. After performing a sub-group analysis of patients with concomitant ulnar styloid fractures, we found that a higher incidence of positive TFCC grind test was seen distal radius fractures with larger ulnar styloid fragments. Specifically, if the size of a concomitant ulnar styloid fragment on pre-operative radiographs is measured to be 7.3mm and above, clinicians should perform a TFCC grind test to rule out TFCC injuries. This is supported by the study from Kim et al, where they reported that tears of TFCC foveal insertion occur in 88-89% of distal radius fractures with concomitant ulnar styloid fractures19. They also found that complete tear of the TFCC foveal insertion, independent of location of the ulnar styloid fracture, was associated with DRUJ instability, contrary to our finding where there was no difference in DRUJ instability. Unfortunately, our study did not include magnetic resonance imaging (MRI) scans or arthroscopic assessment to establish the diagnosis of any TFCC injuries. This is the first clinical study examining the effects of the size of an ulnar styloid fracture fragment on ulnar-sided wrist pain in surgically managed distal radius fractures, and further studies can examine this parameter instead of the anatomical location of an ulnar styloid fracture.In our study cohort, the presence of a concomitant ulnar styloid fracture did not result in a difference in outcome after surgical fixation of distal radius fractures, with both groups have similar ulnar sided symptoms, wrist range of motion and DASH scores up to two years post-operatively. DASH score was the only parameter which had a poorer mean result in Group 0, but this did not achieve statistical significance and a larger study may be required to examine this discrepancy. ECU tendinitis was the only parameter which had a trend towards statistical significance, and this could be related to the fibrous union of ulnar styloid fracture fragments as most did not achieve bony union on follow-up radiographs. Okoli et al documented similar results in terms of Quick Disabilities of the Arm, Shoulder, and Hand (QDASH) and Patient-Rated Wrist Evaluation (PRWE) scores but did not include radiological assessment and ulnar sided symptoms in their study4, and when the base of the ulnar styloid is involved, the concern lies in the association with disruption of the triangular fibrocartilage complex, as well as distal radioulnar joint (DRUJ) instability21. Ulnar-sided wrist pain in the group without an ulnar styloid fracture was similar to the group without and this could be explained by the presence of DRUJ instability despite an intact ulnar styloid. Thus, the importance of an ulnar-styloid fracture likely lies in its possible association with important soft tissue structures contributing to wrist stability, which likely account for much of the morbidity associated with ulnar styloid fractures. Merely fixing the bony structure itself does not guarantee success. It is also noteworthy TFCC tears and DRUJ instability can occur in the absence of ulnar-styloid fractures. Similar findings were found in a study by Lee et al where patients who underwent distal radius fixation without concomitant ulnar styloid fractures had ulnar sided wrist pain, and also had either positive MRI scan findings or intra-operative stress tests, suggesting DRUJ instability22. A careful clinical assessment is critical pre-operatively, with due consideration of either performing pre-operative MRI scans or intra-operative wrist arthroscopy in distal radius fractures with ulnar sided symptoms in absence of an ulnar fracture. However, we acknowledge that this could be challenging to discern in the setting of an acute wrist fracture.The ulnar styloid is well established as a strut for soft tissue attachment over the ulnar aspect of the wrist. The base of the ulnar styloid or fovea forms the attachment of the ulnocarpal ligaments, the triangular fibrocartilage complex (TFCC) and the sheath of the extensor carpi ulnaris (ECU). Non-union of ulnar styloid fracture can result in ulnar-sided wrist pain and extensor carpi ulnaris tendinitisWe recognise that our study is limited by its retrospective nature, and application of our results may be limited to external institutions as this study was performed in a single institution. Further, our study did not include any radiological or arthroscopic assessment of the TFCC. However, this study should prompt subsequent randomised control studies with a larger study cohort and controlled confounding factors, to investigate the impact of ulnar styloid fragment size on clinical outcomes in surgically managed distal radius fractures.In conclusion, the presence or size of ulnar styloid fractures in surgically managed distal radius fractures do not compromise clinical and functional outcomes. However, the presence of an USF of more than 7mm is associated with a positive TFCC grind test.


As some of the renin–angiotensin–aldosterone system (RAAS)-dependent mechanisms underlying the cognitive performance modulation could include oxidative balance alterations, in this study we aimed to describe some of the potential interactions between RAAS modulators (Losartan and Ramipril) and oxidative stress in a typical model of memory impairment. In this study, 48 white male Swiss mice were divided into six groups and received RAAS modulators  and a muscarinic receptors inhibitor  for 8 consecutive days. Then, 24 h after the last administration, the animals were euthanized and whole blood and brain tissues were collected. Biological samples were then processed, and biochemical analysis was carried out to assess superoxide dismutase and glutathione activities and malondialdehyde concentrations. In the present experimental conditions, we showed that RAAS modulation via the angiotensin-converting enzyme inhibition (Ramipril) and via the angiotensin II receptor blockage (Losartan) chronic treatments could lead to oxidative stress modulation in a non-selective muscarinic receptors blocker (scopolamine) animal model. Our results showed that Losartan could exhibit a significant systemic antioxidant potential partly preventing the negative oxidative effects of scopolamine and a brain antioxidant potential, mainly by inhibiting the oxidative-stress-mediated cellular damage and apoptosis. Ramipril could also minimize the oxidative-mediated damage to the lipid components of brain tissue resulting from scopolamine administration. Both blood serum and brain changes in oxidative stress status were observed following 8-day treatments with Ramipril, Losartan, scopolamine, and combinations. While the serum oxidative stress modulation observed in this study could suggest the potential effect of RAAS modulation and scopolamine administration on the circulatory system, blood vessels endothelia, and arterial tension modulation, the observed brain tissues oxidative stress modulation could lead to important information on the complex interaction between renin–angiotensin and cholinergic systems. Despite that it is currently accepted that the essential role played by the renin–angiotensin–aldosterone system (RAAS) is in modulating blood pressure ,2,3, it The correlation between RAAS modulation and other neuropsychiatric disorders was also proposed for mild cognitive impairment , ParkinsFurthermore, the implication of oxidative stress in cognitive processes, brain molecular mechanisms, and RAAS was previously described, and we summarize it in As some of the RAAS-dependent mechanisms underlying the cognitive performance modulation could include oxidative balance alterations, as previously showed in the hippocampus of angiotensin-II-intracerebroventricular-treated mice receiving an angiotensin-converting enzyme inhibitor , in thisScopolamine, a cholinergic neurotransmission blocker, causes both a decrease in neurotransmitter levels and the generation of ROS. All these effects can contribute to poor synaptic transmission, the initiation of inflammatory processes, impaired memory, cognitive decline, and finally, neuronal cell death. Activation of AT1 receptors of angiotensin II can generate a series of negative effects such as oxidative stress, with repercussions on cognitive status. The administration of Ramipril (ACEI) and Losartan (ABR) for various cardiovascular conditions can indirectly improve cognitive status due to the reduction in oxidative stress and anti-inflammatory effect.The design of this study was previously described in  as a parThus, for the evaluation of molecular changes in the context of RAS modulation, 48 white male Swiss mice with an average weight of 30 g were selected from the total experimental group and assigned for biochemical evaluation. Six homogenous groups of mice corresponding to the six experimental conditions, as described in our previous work on the neurobehavioral evaluation , were asDuring the full habituation period, experimental procedures, and tissue sampling, the animals were constantly supervised and evaluated by specialized personnel. Efforts were made to limit the number of animals used and their suffering. Romanian Laws on the animal experimentation in biomedical research and on animal health and welfare and the European Community Guidelines (Directive 2010/63/EU) were considered as gold standard in procedural guidelines of all the activities described in this study. Internal regulations of the study host institution  were also used as procedural guidelines in research, in accordance with the local ethics decision (from 6 October 2016).All the treatments and doses were carefully selected using the minimal effect rule while consulting the pharmaceutic recommendations and scientific literature previous reports on the neurobehavioral and molecular response of RAS modulation and scopolamine administration, as described in ,21. DetaBiological samples—whole blood and brain tissues—were collected from the 48 animals at 24 h following the last chronic administration. Blood samples were allowed to form clots (by air exposure for 2½ h). Then, the clotted-blood-containing tubes were centrifuged at 3500 rpm for 15 min. Blood serum was separated into new tubes, aliquoted, and stored at −20 °C until further biochemical analysis. Brain tissues were extracted from the mice skulls, washed with saline, and used to obtain enzymatic extracts, as described by . Then, 0Total superoxide dismutase specific activity was determined using a Sigma Aldrich commercial 19160 SOD Determination Kit  according to the manufacturer’s instructions. SOD activity was indirectly measured using spectrophotometric based on the water-soluble formazan dye resulting from the water-soluble tetrazolium salt reaction with superoxide anion. Cellular glutathione peroxidase activity was determined using a Sigma Aldrich commercial kit  also by indirect determination based on the measurable nicotinamide adenine dinucleotide phosphate reaction media concentration decrease . The specific enzymatic activity of SOD and GPx was calculated by reference to the total soluble protein content . Malondialdehyde concentrations were assessed using the thiobarbituric acid-reactive substances (TBARs) determination method, as described before ,23. Optit-test were performed for intergroup comparison. Intragroup comparison was verified for homogeneity using the Shapiro–Wilk normality test. Post-hoc analysis was performed using Tukey’s HSD test and Bonferroni corrected t-test. Statistical correlations were assessed using Pearson’s correlation and Spearman correlation. The results were expressed as means ± SEM and the intergroup differences and correlations were regarded as statistically significant when p < 0.05.All numerical data obtained following biochemical evaluation calculus were statistically analyzed using a Minitab 19 statistical tool . One-way analysis of variance (ANOVA) and one/two tailed p = 0.05, one tailed t-test). Despite that, our results showed no significant differences as a result of LOS and RAM treatments, as compared to the control group (p = 0.081). Furthermore, it was observed that both RAM and LOS treatments were not able to prevent SCO effects on the total SOD specific activity . The total SOD specific activity in the blood serum of the animals was decreased when SCO was administered, as compared to the control group  and LOS treatment (p < 0.013) (p = 0.00077). A similar trend was observed following SCO administration in LOS and RAM pre-treated mice, as compared to the control group and respective positive controls, but with a lesser extent in RAM’s case . However, our data showed that RAM pre-treatment was better in modulating brain SOD specific activity in SCO-treated mice, as compared to the injury positive control (SCO), and LOS . On the other hand, the analysis of the data obtained from brain tissues revealed that the total SOD specific activity revealed that RAM treatment could significantly increase total SOD specific activity, as compared to the control group (p = 0.0047). We found that both RAM and LOS treatments could lead to increased cellular GPx specific activity, as compared to the control group . The pre-treatment with RAM in SCO-injured mice significantly prevented the impairment of GPx specific activity . Similarly, our results showed that RAM pre-treatment could better modulate GPx activity in SCO-treated mice, as compared to LOS pre-treatment .Significant differences of the GPx specific activity in the brain tissues were obtained . Malondialdehyde concentrations within the biological samples were evaluated as a biomarker of lipid peroxidation . We obsep = 0.043), and had a suggestive trend as compared to the injury-positive control , leading to the assumption that the angiotensin II receptors blockage could prevent SCO-induced lipid peroxidation traceable from the blood sera. However, we noticed that systemic MDA concentrations were significantly decreased following SCO administration in LOS pre-treated mice, as compared to the control group , while RAM was better at preventing SCO-induced lipid peroxidation . Similarly, we observed that concomitant RAM and SCO exposure lead to significantly decreased MDA levels, as compared to the control group .Regarding the brain lipid peroxidation, we observed that both RAM and LOS chronic treatments improved oxidative status, as suggested by the decreased MDA concentrations, as compared to the control group (p < 0.001) and moderately negative   . In the present experimental conditions, we showed that RAS modulation via the angiotensin-converting enzyme inhibition (RAM) and via the angiotensin II receptor blockage (LOS) chronic treatments could lead to oxidative stress modulation in a non-selective muscarinic receptors blocker (SCO) animal model. In this context, our previous studies showed that both RAM and LOS could lead to significant changes in neurobehavioral landscape of animal models with or without muscarinic receptors modulation. In this way, we showed that RAM and LOS significantly enhanced short- and long-term memory , as wellOur previous studies showed that chronic SCO administration leads to systemic and brain oxidative stress promotion in a typical rodent model ,24. HoweThis study also brings reasonable evidence that both RAS and non-selective muscarinic receptors modulation could lead to oxidative balance modulation. Thus, our results suggested that systemic and brain oxidative status modulation could be obtained through angiotensin-converting enzyme inhibition (RAM administration), angiotensin II receptors blockage (LOS administration), non-selective muscarinic receptors blockage (SCO administration), and their combination. In this experimental context, we observed that the different variants of RAS modulation could boost or reduce the cholinergic receptors blockage effects. The interaction between RAS and the cholinergic system was previously described by  in ovineSeveral recent studies showed that RAM and LOS could exhibit antioxidant properties ,30. Our Regarding the LOS facilitatory effect against oxidative stress, we observed that oxidative modulation was significant in brain tissues, as compared to blood serum, in our animal model. In the cardiovascular system, LOS could prevent angiotensin II binding to the subsequent receptor resulting in arterial pressure decrease and blood vessels constriction . Due to Our results also suggested that RAS system modulation via angiotensin II receptors inhibition could prevent oxidative stress damages occurring in scopolamine-induced neurodegeneration. De-Hua He et al.  extensivSimilarly, a mild antioxidant effect of RAM was suggested by our results. RAM physiologic effects include the inhibition of angiotensin II converting enzyme and the inhibition of lipid peroxidation via antioxidant enzymatic defense potentiation (SOD enzyme activity modulation) . Our resA potential limitation of this study should be addressed: the pro-oxidant effects of scopolamine were seemingly in an incipient stage and not very relevant, which may be the result of the relative short scopolamine exposure duration given the low dosage of 0.5 mg/kg. Additional assays of catalase or other antioxidant enzymes activity might bring clarity to the oxidative dynamics and should be considered in future studies. Immunohistochemical analysis of different neuronal markers, RT-PCR, and western blot of ACE and AT2 could be performed in similar treated groups to provide a clearer perspective on the oxidative status. However, memory impairment in the present animal models was behaviorally confirmed in vivo in our previous preliminary study , which sRAS modulation and non-selective cholinergic receptors blockage could lead to oxidative status modulation. Both blood serum and brain changes in oxidative stress status were observed following 8-day treatments with RAM, LOS, SCO, and combinations. While the serum oxidative stress modulation observed in this study could suggest the potential effect of RAS modulation and SCO administration on the circulatory system, blood vessels endothelia, and arterial tension modulation, the observed brain tissues oxidative stress modulation could lead to important information on the complex interaction between renin–angiotensin and cholinergic systems. Our results showed that LOS could exhibit a significant systemic antioxidant potential partly preventing the negative oxidative effects of SCO and a brain antioxidant potential, mainly by inhibiting the oxidative stress-mediated cellular damage and apoptosis. RAM could also minimize the oxidative-mediated damage to the lipid components of brain tissue resulting from SCO administration.


Coronary no-reflow (CNR) is a frequent phenomenon that develops in patients with ST-segment elevation myocardial infarction (STEMI) following reperfusion therapy. CNR is highly dynamic, develops gradually (over hours) and persists for days to weeks after reperfusion. Microvascular obstruction (MVO) developing as a consequence of myocardial ischemia, distal embolization and reperfusion-related injury is the main pathophysiological mechanism of CNR. The frequency of CNR or MVO after primary PCI differs widely depending on the sensitivity of the tools used for diagnosis and timing of examination. Coronary angiography is readily available and most convenient to diagnose CNR but it is highly conservative and underestimates the true frequency of CNR. Cardiac magnetic resonance (CMR) imaging is the most sensitive method to diagnose MVO and CNR that provides information on the presence, localization and extent of MVO. CMR imaging detects intramyocardial hemorrhage and accurately estimates the infarct size. MVO and CNR markedly negate the benefits of reperfusion therapy and contribute to poor clinical outcomes including adverse remodeling of left ventricle, worsening or new congestive heart failure and reduced survival. Despite extensive research and the use of therapies that target almost all known pathophysiological mechanisms of CNR, no therapy has been found that prevents or reverses CNR and provides consistent clinical benefit in patients with STEMI undergoing reperfusion. Currently, the prevention or alleviation of MVO and CNR remain unmet goals in the therapy of STEMI that continue to be under intense research. Historical records of no-reflow are difficult to track because vascular events developing in early experimental ischemia/reperfusion models, later known as no-reflow, were described well before the no-reflow phenomenon was recognized. The term “no-reflow phenomenon” was coined in 1967 by Guido Majno et al. . Using eThe early records of no-reflow in humans remain elusive or subject to interpretation. It is highly plausible that no-reflow could have played a role in the genesis of ischemic muscular contractures  after surgical embolectomy of arterial thrombi. In 1934, Jefferson  describeMicrovascular obstruction (MVO) is the underlying pathophysiological mechanism of CNR. MVO and CNR after reperfusion of an occluded coronary artery are explained by a joint action of at least four factors: myocardial ischemia, spontaneous or iatrogenic distal embolization, reperfusion-related injury and individual susceptibility (predisposing conditions that increase the odds of developing MVO and CNR). Pathophysiological mechanisms of MVO and CNR are shown in Coronary microcirculation refers to blood circulation in vessels <200 µm in diameter that are not visualized on coronary angiography. It consists of arterioles, capillaries and venules. All 3 structures participate in the MVO and CNR following ischemia and reperfusion, albeit with different roles. Coronary arterioles have a relatively thick smooth muscle wall, act as resistance vessels and are responsible for keeping a constant precapillary pressure of ~45 mmHg in the setting of autoregulation. In the setting of ischemia/reperfusion injury arterioles contribute to MVO and CNR through impaired vasomotor tone (impaired endothelium dependent vasodilation) and propensity to in situ thrombus formation. Morphometric analyses have shown that there are approximately 2200 capillaries per square millimeter in adult human hearts . It has Total cessation or drastic reduction (>80%) of coronary blood flow results in severe myocardial ischemia in subtended myocardium. Previous studies that have investigated cardioprotective measures in ischemia/reperfusion models were potentially conceptually flawed in that they focused on the cardiomyocytes paying little attention to microcirculation. This could be one reason of the failure of cardioprotective measures to translate into clinical benefit . MicrociEndothelial cells are more resistant to ischemia than surrounding cardiomyocytes and may survive hypoxia for minutes to days following the installation of ischemia ,28. EndoLack of high-energy phosphates leads to severe perturbations in ionic hemostasis in endothelial cells. Apart from further contributing to endothelial cell swelling and intracapillary space obstruction as a response to ischemia, altered ionic hemostasis has other negative actions promoting endothelial cell dysfunction. Ischemic endothelial cells show increased concentration of calcium in cytoplasm, which activates endothelial cell contractile elements . Actin fGlycocalyx is perhaps the earliest microcirculation component that is damaged in the course of ischemia/reperfusion. Glycocalyx is an important component of endothelial barrier. Glycocalyx represents a 0.5 µm thick carbohydrate-rich matrix that covers the endothelium surface throughout the capillary system . The thiPlatelets and neutrophils are recruited in the capillaries following myocardial ischemia and contribute to ischemic injury, MVO and CNR . Followi7 pericytes/cm3, pericyte is the second most frequent nonmyogenic cell found in the heart in vitro [Growing evidence suggests that pericytes play an important role in the genesis of CNR. With a density of approximately 3.6 × 10in vitro . Pericytin vitro . Pericytin vitro . In mousin vitro . Cardiacin vitro . In rat in vitro . Recent in vitro ,84. Ischin vitro . It has in vitro .Myocardial ischemia sets into operation a large number of systemic and local vasoconstrictor stimuli that impair the coronary vasodilator reserve and increase the vasoconstrictor tone of microcirculation, which may be alleviated by vasodilator drug therapy . ExperimIn aggregate, myocardial ischemia leads to various alterations affecting all components of microcirculation leading to various degrees of MVO and CNR. Endothelial cells, however, are relatively resistant to ischemia and may remain ultrastructurally intact up to 6 h of ischemia in anesthetized cats . In rat Distal embolization of atherothrombotic fragments from the atherosclerotic plaque occurs spontaneously or during the primary PCI procedure as a result of guide-wire passage, lesion preparation and stent implantation. Angiographically visible distal embolization is documented in 11% to 17% of primary PCI procedures in patients with STEMI ,107,108.Endothelial cells and microcirculation are extremely sensitive to reperfusion-related injury—a condition first described by Jennings et al.  in 1960 Experimental studies in dogs by Kloner et al.  showed tOne of the earliest events that develop following blood flow restoration to ischemic myocardium is exacerbation of ischemia-initiated interstitial and cellular edema. An experimental study in dogs undergoing a 90 min balloon occlusion of left circumflex artery followed by 60 min reperfusion showed increased wall thickness in the reperfused myocardium due to tissue edema eventually leading to CNR because of mechanical compression . CMR ima+/H+ exchanger and Na+/HCO3- symporter leading to proton extrusion from the cells, rapid normalization of intracellular pH, massive Na+ influx, and intracellular Ca2+ overload [2+ in endothelial cells leads to cellular retraction and intercellular gap formation and blebbing resulting in increased vascular permeability and obstruction of intracapillary space. In addition to cellular swelling, increased ATP availability upon restoration of blood flow, restored intracellular pH and abundant cytoplasmic Ca2+ favor the hypercontracture of cardiomyocytes and contraction band formation—a histological marker of reperfusion [2+ content [Ischemia-initiated endothelial cell edema is further accentuated by reperfusion. Upon restoration of blood flow, the extracellular pH is rapidly restored, which stimulates the Naoverload ,124. Theerfusion  further  content ,139. The content ,124.Blood cells contribute to reperfusion-related microvasculature injury and MVO. Serial CMR imaging studies in pigs have shown that interstitial edema is maximal immediately after reperfusion, whereas the maximal content of neutrophils, macrophages, and collagen is observed at 24 h, 4 days and 7 days after reperfusion . NeutropIntramyocardial hemorrhage is one of the most severe manifestations of reperfusion-related injury that is closely linked with MVO and CNR ,141. ExpIschemia/reperfusion is associated with a strong inflammatory response in the infarct zone, predominantly mediated by neutrophils, which contributes to MVO and CNR. Monocytes, macrophages and lymphocytes do participate in the inflammatory response, as well. One experimental study in mongrel dogs, in which a segment of a large epicardial coronary artery was deprived of blood flow for 3 h followed by reperfusion, showed an influx of neutrophils within the media of ischemic/reperfused vessels but not in the nonischemic vessels. Electron microscopic analysis showed that neutrophils were often located between the endothelial cells and the elastic lamina in the ischemic/reperfused vessels . The delOf all mechanisms and mediators proposed to date to explain microcirculation damage in the setting of ischemia/reperfusion, ROS and MMPs have received most attention for their role in the genesis of MVO. Excess production of ROS and activation (or overexpression) of MMPs are underlying mechanisms of tissue damage (including microcirculation) during ischemia/reperfusion and their actions appear to be mutually dependent. In the setting of ischemia/reperfusion, ROS appear to have multiple cellular sources  and the main producers at the molecular level are xanthine oxidase, NADPH oxidase, mitochondrial electron transport chain and uncoupled NO synthase ,98. ExceCNR is highly dynamic in nature, develops gradually (over hours) following coronary blood flow restoration and persists over days to weeks depending on severity, duration and extent of myocardial ischemia and application of therapeutic measures aiming to prevent or alleviate ischemia/reperfusion injury. The diagnostic yield of any method used to diagnose CNR depends on the extent and severity of CNR and the timing of examination. Transient slowing of restored myocardial blood flow or small under-reperfused myocardial segments may go undetected whereas fixed MVO developing over an extensive myocardial area/volume is more reliably detectable. The use of diagnostic methods early (before CNR has developed) or late (after CNR has resolved) after the restoration of epicardial blood flow may fail to detect (or underestimate) CNR. These factors as well as the sensitivity of the method per se used to diagnose CNR may explain the wide variations in the frequency of CNR across the studies. Before analyzing specific methods used to diagnose CNR in clinical setting, two concepts may need clarification. First, the term CNR is used to describe coronary blood flow stasis or MVO after all PCI procedures including elective PCI in patients with chronic coronary syndromes. However, since CNR is an ischemia/reperfusion syndrome resulting from the sequence of coronary artery occlusion (acute ischemia) and reopening (blood flow restoration-related reperfusion injury), blood slowing detected immediately after elective PCI in patients without an acute antecedent (or ongoing) myocardial ischemia may not be CNR. In this scenario, no-flow may be caused by clogged microvasculature by distal embolization of atherosclerotic/thrombotic material in the course of PCI or pre-existing MVO. Second, the definition of CNR requires restoration of blood flow without flow-impeding obstacles at the large coronary artery level. Thus, massive and angiographically visible distal embolization of atherosclerotic/thrombotic material occurring during the primary PCI procedures simply shifts the mechanical obstacle to blood flow from the epicardial artery to a more distal location and, as such it may not represent a true CNR phenomenon.CNR after primary PCI may be diagnosed using invasive (coronary angiography and catheter-based coronary physiology measurements) and noninvasive  methods. Diagnostic methods used to detect CNR in the clinical setting differ widely with respect to their sensitivity to detect the condition and the most optimal timing of their use to diagnose CNR remains unknown.Coronary angiography is routinely used to diagnose CNR following PCI. There are at least four angiographic metrics that have been used to diagnose CNR: Thrombolysis in Myocardial Infarction (TIMI) flow grade, corrected TIMI frame count, myocardial blush grade and TIMI myocardial perfusion grade. TIMI flow grade estimates blood flow in the epicardial coronary arteries. Blood flow is quantified using a scale between 0 and 3 and a TIMI flow grade of <3 is used to diagnose CNR. TIMI flow grade has prognostic value and failure to restore a TIMI flow grade of three was independently associated with increased risk of mortality after PCI in patients with acute coronary syndromes (ACS) . CNR hasMyocardial blush grade or the degree of myocardial contrast staining following intracoronary contrast injection is also used to assess myocardial microvasculature and tissue reperfusion after primary PCI . MyocardTIMI myocardial perfusion grade is also used to assess myocardial reperfusion based on the densitometry of contrast entry, duration, and clearance from the ischemic myocardium . It is sMCE uses gas-filled microbubbles, which are very effective in scattering of ultrasound and an ideal tracer of microcirculation. Microbubbles have rheological properties similar to the red blood cells and their size (<5 μm) allows them to pass through capillaries without blocking them. Microbubbles remain entirely within the vascular space and myocardial contrast intensity following intravenous or intracoronary microbubble injection reflects their concentration in the microvascular compartment of myocardium ,191. TheCMR imaging is the most sensitive technique to detect MVO and diagnose CNR in the clinical setting. By providing multislice views, with a high spatial resolution, CMR imaging enables an accurate quantification and localization of MVO within the infarcted area as well as transmural extent of CNR and necrosis (infarct size) within the infarcted region. CMR imaging also detects the hemorrhagic transformation and extravasation of erythrocytes within the infarct core, which represent a frequent component of CNR that portends a poor prognosis . ContrasSeveral other diagnostic methods have been used to assess CNR in clinical setting. The degree and speed of ST-segment elevation resolution in the post-reperfusion electrocardiogram correlates closely with tissue reperfusion and MVO after primary PCI. A rapid and complete (>70%) ST-segment elevation resolution after primary PCI indicates prompt (and complete) restoration of tissue reperfusion , which iIn aggregate, CNR following reperfusion of patients with AMI should be searched for and diagnosed. Coronary angiography-based techniques are most convenient, but they are performed too early after reperfusion  and, consequently, they underestimate CNR. CMR imaging is the most sensitive technique to diagnose CNR that provides information on its extent and location within the infarcted myocardium in clinical setting. Apart from CNR diagnosis and characterization, CMR imaging also detects intramyocardial hemorrhage and accurately estimates infarct size, further improving the risk stratification of patients with AMI. Other techniques  have limited use for the diagnosis of CNR in current clinical practice.The frequency of CNR after primary PCI differs widely. Aside from sensitivity of the method used to detect CNR and the timing of the assessment , a number of other factors appear to predispose to CNR after primary PCI including, infarct size, pre-existing endothelial and microvascular dysfunction, cardiovascular risk factors, culprit lesion characteristics, invasiveness of coronary intervention and genetic predisposition to CNR.Several experimental ,208 and Pre-existing endothelial and microvascular dysfunction are common in patients with AMI and they likely contribute to MVO and CNR after primary PCI . CoronarCulprit lesion morphology appears to impact on frequency of CNR after primary PCI. Atherothrombotic plaques responsible for ACS are larger and softer  and are more prone to be fragmented (and embolized) during coronary interventions . One stup = 0.059) with intramyocardial hemorrhage [Although diabetes mellitus is associated with generalized endothelial  and micrmorrhage . The assmorrhage  suggestimorrhage . Howevermorrhage . Anothermorrhage . A recenmorrhage . Despitemorrhage , recent morrhage ,255,256.morrhage ,256. A hmorrhage ,258. Premorrhage ,260.There appears to be a genetic predisposition to CNR. A 2007 case-control study showed that survivors of AMI who developed CNR after PCI had more compact fibrin network and resistance to lysis , suggestCNR can be clinically silent or manifest as angina, ST-segment (re)elevation, sudden hemodynamic deterioration while in the catheterization laboratory, malignant ventricular arrhythmias, early congestive heart failure (or cardiogenic shock) and cardiac death . At longLong-term studies have further confirmed a worse long-term prognosis associated with CNR. Morishima et al.  assessedTransient CNR, defined as CNR during the primary PCI procedure in the absence of flow limiting obstructions at the culprit lesion that reverses to normal blood flow at the end of the procedure, is also associated with clinical outcomes ,271,272.The underlying mechanisms of the association of CNR with poor outcomes are unclear. Infarct size—a well-known correlate of poor outcome after primary PCI for STEMI—is a strong correlate of CNR. In this regard, CNR is a manifestation of severe ischemic damage of the myocardium characterized by prolonged ischemia affecting extensive parts of the myocardium. Factors that predispose to CNR  also predispose to a poor clinical outcome, regardless of CNR. Although infarct size is closely related to CNR, whether CNR per se impacts on the infarct size remains controversial. Since, the zone of CNR is almost invariably located within the dead tissue, it has been suggested that CNR does not cause ischemic cell death . ClinicaDespite more than 50 years of research, little progress has been made in finding a treatment strategy of proven efficacy to be routinely used to prevent CNR in patients with STEMI. Many therapies that showed benefits in reducing MVO and CNR in animals failed to result in similar benefits in clinical setting in patients with STEMI. Although some therapies improved some markers of reperfusion, so far, no therapy applied to prevent or alleviate MVO or CNR has resulted in clinical benefit in terms of reduction of hard clinical endpoints such as mortality. All pathophysiological mechanisms of CNR—distal embolization, myocardial ischemia and reperfusion-related injury—and predisposing factors (when possible) have been targeted by nonpharmacological or pharmacological preventive strategies as single or combined strategies. These therapies were applied before, during or after cardiac catheterization and primary PCI procedure to prevent or alleviate CNR (when it develops).Nonpharmacological and pharmacological therapies have been used to reduce the incidence of distal embolization in the setting of primary PCI procedures in patients with STEMI. Intuitively, direct stenting (stenting without predilation) was expected to reduce MVO and CNR by reducing distal embolization. Early randomized studies ,280 withDistal protection devices have been used to reduce distal embolization of atherothrombotic debris during the primary PCI. The EMERALD  trial randomized 501 patients with STEMI to PCI plus balloon occlusion and aspiration distal microcirculatory protection system or PCI alone. Distal protection system failed to reduce CNR and had no significant impact on final TIMI flow, final corrected TIMI frame count, myocardial blush grade, ST-segment resolution > 70%, infarct size or 6-month incidence of major adverse cardiovascular events (MACE) . In othep = 0.28). However, it showed a significant increase in the incidence of stroke with aspiration thrombectomy [Mechanical or aspiration thrombectomy devices have also been used to reduce distal embolization in the setting of primary PCI. A 2013 meta-analysis showed that aspiration thrombectomy reduced the risk of MACE and all-cause mortality with no impact on final infarct size or LVEF. With respect to markers of tissue reperfusion, aspiration thrombectomy improved ST-segment resolution and TIMI blush grade. Mechanical thrombectomy did not improve TIMI blush grade, infarct size or the incidence of MACE; however, it improved ST-segment resolution at 60 min . The metmbectomy . These sp = 0.51) [A strategy of deferred stenting—a two-step strategy of initial reperfusion by balloon angioplasty  followed by stent implantation hours or days thereafter—was used with the hope that it would reduce the rate of vessel dissection or distal embolization. In a study of 101 patients with STEMI with ≥1 factor for CNR, deferred stenting reduced the frequency of CNR (2% vs. 14%) and improved myocardial salvage assessed by CMR at 6 months . However = 0.51) . The treMesh-covered stents have been developed to prevent distal embolization by trapping and excluding embolism-prone material at the level of culprit lesion in patients with STEMI. The MASTER (Safety and Efficacy Study of MGuard Stent After a Heart Attack) trial, which randomized 433 patients with STEMI to a mesh-covered stent (MGuard stent) or a bare-metal or drug-eluting stent, showed that the mesh-covered stent improved complete ST-segment resolution ≥ 70% and postprocedural TIMI flow grade of three with no significant impact on infarct size assessed by CMR or 30-day incidence of MACE . Patientp = 0.013) [p = 0.004), improved left ventricular end-diastolic volume at 6 months but had no effect on the MACE rate at 1 year compared with intravenous tirofiban [Glycoprotein 2b/3a receptor inhibitors were used to reduce thrombotic events including distal embolization and improve microcirculation due to their inhibitory effects on platelet aggregation. However, clinical results with these agents have not been convincing. In the On-TIME-2  trial, routine prehospital high-bolus dose of tirofiban improved ST-segment resolution before and one hour after PCI . The tri= 0.013) . A recenirofiban . In the irofiban . The AIDirofiban . The CMRirofiban . Glycoprirofiban . Currentirofiban .In summary, therapy against distal embolization has failed to produce beneficial clinical effects in a consistent manner. Although the reasons why this therapy failed to prevent or ameliorate CNR are unclear, two putative mechanisms may be offered: first, therapy  may fail to prevent distal embolization or even may facilitate it at the time of device placement, and second, distal embolization may play a modest role in the pathophysiology of MVO and CNR.+/H+ ion exchanger inhibitors (cariporide), Na+/Ca2+ ion exchanger inhibitors , K+ATP channel agonists (nicorandil), atrial natriuretic peptides, erythropoietin, adenosine, endothelin receptor inhibitors, protein kinase C inhibitors (delcasertib) as well as therapeutic strategies of hypothermia, hyperoxemia and ischemic conditioning have been used to prevent MVO and CNR [A large number of pharmacological agents or therapeutic strategies, alone or in combination, have been used to reduce infarct size, boost myocardial salvage and prevent or reduce reperfusion injury and MVO or CNR after primary PCI in patients with STEMI. Apart from antithrombotic  and anticoagulant  drugs that are routinely used during the primary PCI, numerous drugs including, statins, angiotensin-converting enzyme inhibitors, calcium channel blockers, beta-blockers, antioxidants , neutrophil and complement system inhibitors (pexelizumab), antidiabetic drugs (exenatide), glucose–insulin–potassium infusion, antiischemic agents (trimetazidine), inhibitors of mitochondrial permeability transition pore opening (cyclosporine), NO donors, Na and CNR . The mosAdenosine—a purine nucleoside and a potent vasodilator of arterioles and microcirculation via binding to A2 receptors—has been tested as a means to prevent (or reverse) MVO and CNR after primary PCI. Experimental studies suggested that adenosine ameliorates ischemia/reperfusion injury, limits infarct size and improves left ventricular function. Earlier studies showed that intravenous adenosine reduces infarct size when used as adjunct to thrombolytic drugs or primary PCI although with a neutral effect on clinical outcomes in patients with STEMI ,304. Thep = 0.75 for nitroprusside vs. saline) [Sodium nitrite—a NO donor—is a powerful arteriolar vasodilator with antiplatelet and inti-inflammatory properties. The Nitrates in Acute Myocardial Infarction (NIAMI) trial randomized 229 patients with STEMI to receive either an intravenous infusion of 70 μmol sodium nitrite or matching placebo. The drug failed to reduce the infarct size measured by CMR at 6–8 days. In addition, there were no significant differences between nitrite and placebo with respect to the area under the curve of troponin I and creatine kinase, left ventricular volumes or ejection fraction measured at 6–8 days and infarct size measured at 6 months . In the  saline) . Based oCalcium channel blockers  have been used to treat CNR, but the quality of studies is poor . A 2015 ATP channels [The impact of beta-blocking agents on MVO and infarct size in patients with STEMI remains controversial. One study in Yorkshire pigs showed that intravenous metoprolol was associated with a 5-fold higher myocardial salvage and a significant improve in LVEF compared with placebo . This efchannels . Anotherchannels . The Effchannels . Howeverchannels . These dStatins were thought to improve microcirculation and prevent or treat CNR through their pleiotropic effects including endothelium protection, microcirculation dilatation and antithrombotic and anti-inflammatory actions. The Efficacy of High-Dose AtorvaSTATIN Loading Before Primary Percutaneous Coronary Intervention in ST-Elevation Myocardial Infarction (STATIN STEMI) trial showed that high-dose (80 mg) atorvastatin pretreatment improved angiographic MVO  and ST-segment resolution but not MACE at 30 days compared with low (10 mg) atorvastatin dose . In the Intracoronary fibrinolysis has been used to prevent or treat MVO and CNR. In the Trial of Low-dose Adjunctive alTeplase During prIMary PCI (T-TIME), intracoronary alteplase at doses of 10 mg and 20 mg did not reduce MVO compared with placebo . HoweverIntracoronary epinephrine can mediate coronary vasodilatation at lower doses and, consequently, has been used mostly to reverse CNR. Navarese et al.  tested iSuboptimal platelet response to dual antiplatelet therapy was associated with a greater extent of MVO . This haATP channel opener and nitrate—reduced the incidence of TIMI flow grade ≤ 2 by 37% and improved LVEF by 3.7% with no impact on peak creatine kinase. A multicenter, randomized, double-blind, dose-finding placebo-controlled study of ITF-1697—a C-reactive protein-derived tetrapeptide—showed no difference between 0.1 and 1.0 μg/kg/min study arms or placebo with respect to post-procedural TIMI flow, corrected TIMI frame count, blush grade and ST-segment resolution in 402 patients with AMI [Over the years, a plethora of drugs and strategies, alone or in combination, have been used to protect microcirculation and prevent MVO or CNR and reduce infarct size in patients with STEMI. Cyclosporine A—an inhibitor of mitochondrial permeability transition pore opening—reduced infarct size in a small, randomized study of 58 patients with STEMI undergoing primary PCI . Howeverwith AMI . In a rawith AMI . The impwith AMI .p = 0.32) [Earlier studies with small numbers of patients suggested that ischemic postconditioning may reduce the infarct size estimated by CMR ,354. How = 0.32) . RepreseIn summary, despite decades of intensive research and testing of numerous agents and therapeutic strategies targeting all known pathophysiological mechanisms of MVO and CNR, no satisfactory therapy has been found to prevent or reverse these phenomena and consistently improve the clinical outcome. Although there is no evidence to support the routine use of any above-mentioned therapeutic strategies during primary PCI, in patients with large thrombus burden at high risk for CNR, manual thrombus aspiration with direct stenting continues to be used to reduce distal embolization. Vasodilator therapy has been the mainstay of CNR therapy for almost 50 years in patients with AMI who develop persistent CNR and continues to be used in catheterization laboratories based on operator’s personal experience or intuition, albeit without evidence of a proven benefit. Even though vasodilator therapy may improve some markers of reperfusion (particularly angiographic markers), it has largely failed to reduce infarct size or improve clinical outcome. Powerful antithrombotic drugs like glycoprotein 2b/3a may be used but they increase the risk of intramyocardial hemorrhage.MVO and CNR are relatively frequent phenomena that develop following reperfusion therapy in patients with STEMI. The frequency of CNR or MVO after primary PCI differs widely depending on the sensitivity of diagnostic methods and timing of examination. Coronary angiography is readily available and most convenient to diagnose CNR but it is highly conservative due to suboptimal (too early) timing of examination (end of PCI procedure) and low sensitivity of angiographic criteria. CMR imaging is the most sensitive method to diagnose MVO and CMR that provides information on the presence, localization and extent of MVO. CMR imaging detects also intramyocardial hemorrhage and accurately estimates infarct size in clinical setting. MVO and CNR markedly negate the benefits of reperfusion therapy and contribute to poor clinical outcomes including adverse remodeling of left ventricle, worsening or new congestive heart failure and increased risk of mortality. Since MVO and CNR are important predictors of clinical outcome, patients with STEMI developing these phenomena are in need of careful risk stratification and tailored therapy. Despite almost 50 years of research and targeting of all known factors (and mechanisms) involved in the pathophysiology of MVO and CNR, no therapy has been found that prevents or reverses these phenomena (when they develop) and provides consistent clinical benefit in patients with STEMI. While no MVO or CNR preventive therapies can be recommended to be routinely used in patients with STEMI undergoing primary PCI, early reperfusion by primary PCI remains the only means to stop ongoing ischemia, reduce the infarct size and consequently reduce the occurrence and extent of MVO and CNR. For the time being, prevention or alleviation of MVO and CNR remain unmet goals in the therapy of STEMI that continue to be under intense research.


Domestic cats are popular companion animals, however not all live in human homes and many cats live within shelters or as free-roaming, unowned- feral or stray cats. Cats can transition between these subpopulations, but the influence of this connectivity on overall population dynamics, and the effectiveness of management interventions, remain poorly understood. We developed a UK-focused multistate Matrix Population Model (MPM), combining multiple life history parameters into an integrated model of cat demography and population dynamics. The model characterises cats according to their age, subpopulation and reproductive status, resulting in a 28-state model. We account for density-dependence, seasonality and uncertainty in our modelled projections. Through simulations, we examine the model by testing the effect of different female owned-cat neutering scenarios over a 10-year projection timespan. We also use the model to identify the vital rates to which total population growth is most sensitive. The current model framework demonstrates that increased prevalence of neutering within the owned cat subpopulation influences the population dynamics of all subpopulations. Further simulations find that neutering owned cats younger is sufficient to reduce overall population growth rate, regardless of the overall neutering prevalence. Population growth rate is most influenced by owned cat survival and fecundity. Owned cats, which made up the majority of our modelled population, have the most influence on overall population dynamics, followed by stray, feral and then shelter cats. Due to the importance of owned-cat parameters within the current model framework, we find cat population dynamics are most sensitive to shifts in owned cat husbandry. Our results provide a first evaluation of the demography of the domestic cat population in the UK and provide the first structured population model of its kind, thus contributing to a wider understanding of the importance of modelling connectivity between subpopulations. Through example scenarios we highlight the importance of studying domestic cat populations in their entirety to better understand factors influencing their dynamics and to guide management planning. The model provides a theoretical framework for further development, tailoring to specific geographies and experimental investigation of management interventions. Felis catus) is one of the most popular companion animal species globally. In the UK alone, there are over 10 million owned cats  and vary per scenarios 1 [S1] and 2 [S2] are also included.Probabilities were converted into an approximate monthly rate using the formula p~1-(1-p)(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.