Bird related hypersensitivity pneumonitis (HP) is becoming more common than other forms of HP around the world. We present two cases of HP, associated with exposure to visiting birds which had nested within their homes in semi urban areas of Colombo, Sri Lanka. A 65-year-old female (case 1) and a 61-year-old male (case 2) presented to the chest clinic complaining of gradually progressive and persistent chronic dry cough and dyspnoea during the year 2018. Both were found to have close contact with red-vented bulbuls (Konda kurulla) in their homes for more than 6 months prior to onset of symptoms and denied any other risk exposures in detail history taking. In both patients, high-resolution computed tomography chest (HRCT) showed centrilobular nodules of ground glass density with significant lobular air trapping. Video-assisted thoracoscopic (VATs) lung biopsy of case 1 showed patchy and focal interstitial thickening with lymphocytic infiltrate, minimal fibrosis, and few noncaseating granulomata within the interstitium. Transbronchial lung biopsy of case 2 showed thickened alveolar septae with lympho-histiocytic infiltrate and occasional neutrophils and eosinopils. Both showed severe reduction in forced vital capacity (FVC) at presentation. Multidisciplinary diagnosis of HP associated with red-vented bulbuls was made. Both achieved good improvement in clinical, lung function, and radiological assessment following removal of offending antigen exposure and treatment with oral corticosteroids. Hypersensitivity pneumonitis (HP) is an immunologically-mediated inflammatory lung disease caused by repetitive inhalation of antigens in a susceptible host –3. PotenBoth patients were retired professionals from semiurban areas in Colombo who denied exposure to any organic or inorganic particles, which can cause HP, within the last two years. However, repeated questioning revealed that there had been close contact with red-vented bulbuls which frequented their houses. The birds nested in chandeliers hung directly above the sofas in their living rooms where these patients spent most of their leisure times. Though there had been debris falling on to the sofa as the birds moved within the nests above, they had not attempted to remove the nests as they enjoyed their presence. The living rooms had been kept closed during the periods when the birds reproduced, to protect the eggs and nestlings. Additionally, both patients had closely handled the nestlings until they fledged. This confirmed that there had been continuous exposure to antigens of red vented bulbuls for around 8–10 hours per day for 10–18 months with a proximity of 2–3 meters in a closed or partially closed environment, in these patients.A 65-year female presented to the clinic with insidious-onset persistent and progressive dyspnoea, cough, and wheezing for six months. She was a diagnosed diabetic and had a history of allergic rhinitis. She revealed close contact with red-vented bulbul birds for around one-and-half years. Erythrocyte sedimentation rate (ESR) was 41 mm/h. Chest radiography showed patchy opacifications in right mid zone and reticular nodular shadows in mid zones of both lung fields . SaturatA 61-year-old male presented with insidious-onset, persistent and progressive dyspnoea and dry cough for four months. At the onset, he had low grade fever and constitutional symptom which subsided over a few weeks. He was a known hypertensive. He revealed close contact with red-vented bulbuls for around ten months. ESR was 78 mm/h. Chest radiography showed reticular nodular shadows in mid and lower zones of both lung fields . SpiromeA diagnosis of subacute HP associated with exposure to red vented bulbuls was made for both cases at a multidisciplinary meeting for interstitial lung diseases held at the National hospital of Sri Lanka. The patient of case 1 achieved good clinical and lung function improvement (FVC 91.9%) by about two months and that of case 2 showed clinical and lung function improvement (FVC 72%) around the third month of treatment with oral prednisolone (0.5 mg/kg) tail down regimen and the avoidance of the offending exposure. Both patients did not have any recurrences following avoidance of exposure to red-vented bulbuls and achieved normal FVC values in spirometry assessment with treatment of oral prednisolone within one-year follow-up period.μm) found in feathers, faeces, and other animal products [Bird related HP (Bird fancier's lung) is increasingly becoming prevalent around the world and shows worse outcome than other forms , 9. Birdproducts .Suspicion of an association between symptoms and contact with a provoking antigen is the first step in the diagnostic process of HP, combined with the measurement of serological markers and specific IgG antibody levels, radiological findings, lung function assessment, bronchoalveolar lavage, and lung biopsy for the complete workup , 12. TheAir space involvement of HP in the lung parenchyma presents as patchy ground glass opacities and/or centrilobular nodules in HRCT . ShuntinProper history plays a key role in HP as early avoidance of causative associations invariably brings complete or partial recovery whereas delay would cause irreversible lung fibrosis. Multidisciplinary approach is highly recommended when the diagnosis is challenging .Pycnonotus cafer) also known as Kondakurulla in Sinhala, is a resident breeder across the Indian subcontinent including Sri Lanka, and has established itself in several Pacific islands, parts of the United Arab Emirates, United States, and possibly New Zealand, not limiting to a part of the world [The red-vented bulbul (he world –19. Theyhe world , 21.To our knowledge, all published cases of bird related HP are associated with birds raised as pets or for farming. An important fact to be highlighted is that when a clinician questions regarding risk exposure to birds, the patient would give a negative answer initially if they have not raised birds purposefully. The opportunity to remove the exposure would be missed in such instances. Hence, the present observation highlights the importance of specific questioning regarding this kind of casual exposures to visiting birds within living places.Birds nesting and breeding inside houses is considered to bring prosperity to the household in some cultural beliefs. Hence updating public on harmful aspects of these associations would also be important.


A 90-year-old male developed acute onset of abdominal and lumbar pain due to the rupture of an 11-cm abdominal aortic aneurysm. A congenital fused pelvic kidney perfused by three renal arteries arising from iliac axes was detected. In an emergent setting, an aorto-uni-iliac endograft was deployed through right femoral surgical access with occlusion of the upper right renal artery. An occluder device was placed in the common iliac artery above the renal artery through left femoral access. A femorofemoral crossover bypass completed the procedure. The patient developed acute renal failure, with no dialysis necessity. One-month computed tomography angiography showed procedure success. Congenital renal abnormalities occur in 3.3 to 11% of the populationIn this population, the number and site of origin of renal arteries are extremely variable and, for this reason, the treatment of AAA is more complex. Assessment of renal arteries  and renal function  is mandatory for optimal planning and to obtain technical and clinical success after any type of procedure .Management of ruptured AAA is challenging and perioperative mortality after emergency open or endovascular treatment reaches 53%.The aim of this article is to report a case of emergency endovascular treatment of a ruptured AAA with congenital fused pelvic kidney (CFPK).A 90-year-old male presented to the emergency department complaining of severe abdominal and back pain. The symptoms began 3 hours earlier. The patient was severely obese and under chronic pharmacological therapy for arterial hypertension and coronary artery disease (previous coronary artery bypass graft). The abdominal computed tomography angiogram (CTA) showed an 11-cm AAA with a retroperitoneal hematoma and a posterior wall rupture . The aoPreoperative planning was performed using 3mensio Vascular  during patient transport to the operating theater and anesthetic induction.Under general anesthesia, inguinal longitudinal cut-down was performed bilaterally and the common femoral arteries were isolated. An Endurant aorto-uni-iliac endograft (Medtronic Endovascular) was introduced through the right access. The C-arm was rotated at 50°-left anterior oblique and the preoperative angiogram was performed. The endograft was deployed below the origin of superior mesenteric artery, using the inframesenteric nondilated aorta as proximal sealing zone (∼20 mm in length). Two Endurant endograft extensions (Medtronic) were necessary to obtain sufficient sealing at the right common iliac artery with the occlusion of the upper renal artery. Through the left access, a Talent occluder was deployed in the proximal left common iliac artery, above the origin of left renal artery. The completion angiogram showed patency of the endografts and no endoleak from left iliac axis or renal or lumbar arteries . The prVentilator support was removed after 24 hours and the abdominal and lumbar pain disappeared. Acute renal failure developed 5 but nAt 1 month, the renal function remained mildly altered (creatinine serum level: 1.9 mg/dL). After intravenous hydration, the abdominal CTA showed patency of the aorto-uni-iliac endograft and femorofemoral bypass graft, sac shrinkage, and no evidence of endoleaks. The pelvic kidney presented no ischemic lesions and was perfused by two renal arteries (lower right and left) .Congenital pelvic kidney (CPK) is defined as the presence of one ectopic kidney low lying in the abdomen.Congenital fused pelvic kidney, also called “pancake kidney,” is a rare congenital anatomic variant consisting of unique lobulated pelvic renal mass of dual parenchymatous systems.Considering patients who underwent major aortic surgery, ∼0.18% presented a CPK.These two anatomical variants are often associated with multiple aberrant renal arteries arising from the aortic bifurcation and iliac arteries. AAA treatment is technically challenging, and has a higher risk of kidney failure.In the current case, endovascular treatment was preferred because of the advanced age and the severe obesity of the patient.No bifurcated endografts were available in our center for procedures in an urgent/emergent setting, so aorto-uni-iliac endografting and femorofemoral bypass were obligatory choices. Considering the sites of origin of the three renal arteries, the coverage of upper right renal artery was inevitable to obtain an adequate distal sealing zone. Also in case of left-side deployment of the aorto-uni-iliac endograft, the upper right renal artery would have been excluded by the occluder device deployment in the right common iliac artery.To reduce the risk of persistent type 2 endoleak, a sac embolization may be intraoperatively performed. This adjunctive procedure was not executed to reduce time of intervention and was not considered strictly necessary. Indeed, no patent lumbar arteries had been detected at preoperatory CTA, and the upper right renal artery arises from a nondilated segment making unlikely a back flow into the aneurysmal sac.In case of renal anomalies, assessment of preoperative CTA is essential to define their number, origin, and size. In the literature, sacrifice of accessory renal arteries with diameter < 3 mm is considered noninfluential on renal function.Several cases of treatment of AAA associated with CPK or CFPK have been reported in literature .8910Surgical treatment of AAA in patients with pelvic kidney (CPK or CFPK) is more complex than in the general AAA population. Perfusion of the kidney must be maintained during aortic clamping and graft sewing. Renal arteries with diameter > 3mm should be reimplantedTotal endovascular treatment has also been described. Morales and GreenbergHybrid treatment has also been reported. In a case of CFPK with renal arteries arising from the AAA, a double iliac-renal bypass was primarily sutured and an aorto-bifurcated endograft was deployed to exclude the aneurysm.In conclusion, in case of CPK or CFPK, endovascular, open surgical, and hybrid treatments of AAA have been described in literature and are potentially feasible. The most suitable treatment must be chosen according to the patient's clinical condition and arterial anatomy. Our case demonstrates that accurate preoperative planning is vital to achieve technical success and to preserve renal function in this complex subset of patients.


Diabetic ketoacidosis (DKA) is a potentially fatal endocrine emergency resulting from uncontrolled diabetes mellitus (DM). The development of DKA has been linked to a number of precipitating factors such as infectious process, ischemia, medications, and other medical-surgical illnesses. These factors have been found to aggravate or unmask pre-existing glucose dysregulation secondary to absolute or relative insulin deficiency and increased levels of counter-regulatory hormones. We describe the case of a 61-year-old male with a history of insulin dependent DM who develops DKA postoperatively after a three-vessel coronary artery bypass surgery and mitral valve repair while in the intensive care unit (ICU). The patient’s postoperative course was complicated by presumed pneumonia and hyperactive delirium. On postoperative day (POD) five, the patient’s insulin infusion was held due to non-symptomatic hypoglycemia. Eleven hours later, the insulin infusion was resumed to treat DKA after laboratory findings revealed hyperglycemia, an elevated β-hydroxybutyrate, and anion gap metabolic acidosis. Multiple contributing factors for the development of DKA are suspected and discussed. It is paramount that clinicians are knowledgeable of the multiple factors that can contribute to the development of DKA in the ICU. Diabetic ketoacidosis (DKA) is a potentially fatal endocrine emergency which acutely develops from precipitating factors which can aggravate or unmask pre-existing glucose dysregulation. In addition to absolute or relative insulin deficiency, increased levels of counter-regulatory hormones  promote hyperglycemia by increasing gluconeogenesis, glycogenolysis and decreasing insulin sensitivity in peripheral tissues. This leads to lipolysis and subsequent elevations in free fatty acids which then undergo hepatic oxidation to ketone bodies (β-hydroxybutyrate and acetoacetate). Accumulation of these ketone bodies results in ketonemia and anion gap metabolic acidosis -3. In up to 40% of DKA cases, known diabetics experienced episodes of extreme physiologic stress secondary to an infectious process, ischemia, or other medical-surgical illnesses. The lack of exogenous insulin due to medical noncompliance, inadequate dosing, and in some cases, malfunction of insulin pumps accounts for an estimated 21% to 49% of DKA cases. Medications that alter carbohydrate metabolism, anorexia, and endocrine disorders have also been implicated as risk factors for DKA. Notably, up to 10% of DKA cases are described as idiopathic . AlthougHere we describe a case of DKA precipitated by multiple factors in the ICU in a known insulin-dependent diabetic patient on postoperative day (POD) five after a three-vessel coronary artery bypass graft (CABG) and mitral valve repair (MVr). Multiple cases of euglycemic DKA developing in the ICU secondary to singular factors such as pregnancy or use of sodium-glucose cotransporter 2 inhibitors have been previously reported in the literature . HoweverA 61-year-old male with a past medical history of a cerebrovascular accident with residual left-sided deficit, hypertension, hyperlipidemia, and type 2 diabetes mellitus (DM) managed on an insulin pump with a perioperative hemoglobin A1c (HbA1c) of 7.2%  underwent a three-vessel CABG and MVr with annuloplasty ring. The patient was weaned off vasopressors by POD four yet continued on a low dose of inotrope. Hydrocortisone was started, empirically, in the immediate postoperative period for circulatory shock presumably due to relative adrenal insufficiency. This therapy was discontinued on the morning of POD five.Given the presence of fever and infiltrates on chest X-ray, cefepime and vancomycin were initiated as empiric coverage for a suspected left lower lobe pneumonia on POD four. Of note, blood cultures remained no growth to date. The patient was also started on oral quetiapine 25 mg twice daily for acute hyperactive delirium. The patient’s blood glucose (BG), prior to quetiapine initiation, ranged from 79-206 mg/dL . Our cardiothoracic surgery (CTS) insulin infusion protocol, with specific blood glucose (BG) goals of 110-140 mg/dL, is typically active until POD two before it is transitioned to sliding scale with or without a scheduled insulin regimen for extubated patients who are able to tolerate a diet. Diabetics that typically manage their BG with an insulin pump are permitted to resume use of this device given they have the cognition to manipulate the settings for insulin administration based on point of care (POC) BG readings. However, this patient’s altered mentation prohibited safe inpatient use of his insulin pump and thus necessitated the continuation of an insulin infusion. Due to the patient's documented dysphagia, enteral nutrition via a nasogastric tube was continued after he was extubated.On the morning of POD five, the insulin infusion was held secondary to non-symptomatic hypoglycemia with a POC BG of 69 mg/dL. Quetiapine therapy was discontinued after the third dose secondary to lethargy. Small doses of intravenous (IV) haloperidol on an as needed basis for agitation were utilized instead. The patient received one dose of IV haloperidol 1 mg during the early evening of POD five and was later started on a low dose dexmedetomidine infusion for persistent agitation. That same evening, the patient developed atrial fibrillation with rapid ventricular response. This was managed with three 150 mg boluses of IV amiodarone along with a short amiodarone infusion , an anion gap of 17 mmol/L, and a lactate of 4 mmol/L thus suggesting a diagnosis of DKA. Enteral feedings were discontinued. DKA was managed with four liters of balanced crystalloids for fluid resuscitation, two boluses of regular insulin IV , and re-initiation of the insulin infusion that had been held for 11 hours given the previous hypoglycemic episode  as contributing factors for DKA manifestation . AlthougSeen as early as four days, AA-induced DKA can occur at any time following the initiation of medication. Risk factors include male gender, age 30-60 years, history of DM, history of schizophrenia, type of AA , polypharmacy, and ethnicity (African or Caucasian descent) while weight gain is an independent variable -8,10. OuAlthough amiodarone-induced DKA has not been reported, hyperglycemia secondary to amiodarone-induced dysregulation of carbohydrate metabolism has been previously described in multiple case reports involving both children and adults. Patients were noted to have normal baseline thyroid studies and no documentation of thyroid dysfunction. Alterations in glucose metabolism were reported to occur as early as five hours after initiation of amiodarone while in some cases manifestations of dysglycemia were delayed up to one year. In all cases, BG normalized after amiodarone was discontinued; only one patient was unable to withdraw from antihyperglycemic therapy -13. One The absence or insufficient provision of insulin therapy is the second most common precipitating cause for DKA -3. AfterOsmotic diuresis secondary to DKA can result in severe electrolyte depletion, particularly hypophosphatemia. In addition, a case series identified a plethora of reasons leading to hypophosphatemia in CTS patients. The inverse relationship between hypophosphatemia and hyperglycemia in non-diabetic patients has been previously documented . It has The development of DKA in an ICU is an unusual phenomenon. Generally, patients are admitted to the ICU for DKA management given the potential complications related to severe cases. This case presents multiple contributory factors for the development of DKA, in the ICU, in a diabetic patient status-post CABG and MVr. Furthermore, this case highlights the fact that DKA can develop in the critical care setting and critical care practitioners need to be vigilant at recognizing these, and other, potential contributory factors for DKA.


TNF), a purely cytotoxic gene for antitumor activity (RGD4C-AAVP-TNF); in the other construct, we delivered Herpes simplex virus thymidine kinase (HSVtk) for in tandem molecular-genetic imaging and targeted therapy (RGD4C-AAVP-HSVtk) utilizing ganciclovir (GCV) for a suicide gene therapy. Both AAVP constructs demonstrated antitumor activity, with damage to the tumor-associated neovasculature and induction of cell death evident after treatment. In addition, the ability to monitor transgene expression with a radiolabeled HSVtk substrate pre and post GCV treatment demonstrated the theranostic potential of RGD4C-AAVP-HSVtk. We conclude that targeted AAVP constructs delivering either cytotoxic TNF or theranostic HSVtk followed by suicide gene therapy with GCV have comparable preclinical efficacy, at least in this standard experimental model. The results presented here provide a blueprint for future studies of targeted gene delivery against human glioblastomas and other brain tumors.Glioblastoma persists as a uniformly deadly diagnosis for patients and effective therapeutic options are gravely needed. Recently, targeted gene therapy approaches are reemerging as attractive experimental clinical agents. Our ligand-directed hybrid virus of adeno-associated virus and phage (AAVP) is a targeted gene delivery vector that has been used in several formulations displaying targeting ligand peptides to deliver clinically applicable transgenes. Here we compared different constructs side-by-side in a tumor model, an orthotopic model of xenograft human glioblastoma cells stereotactically implanted in immunodeficient mice. We have used divergent therapeutic strategies for two AAVP constructs, both displaying a double-cyclic RGD4C motif ligand specific for alpha V integrins expressed in tumor vascular endothelium, but carrying different genes of interest for the treatment of intracranial xenografted tumors. One construct delivered tumor necrosis factor ( Glioblastomas are lethal intracranial tumors of the central nervous system characterized by high morbidity and mortality due to their high tumor cell proliferation rate and marked neovascularization, allowing them to infiltrate crucial structures in the brain . The delcis-genomic elements from adeno-associated virus (AAV) and single-stranded M13-derived bacteriophage displaying tumor-homing ligand peptide motifs to target endothelial and tumor cell surface receptors and to mediate selective internalization of ligand-directed viral particles -FEAU, and/or a suicide gene therapy strategy when combined with ganciclovir (GCV). This theranostic convergence of molecular-genetic imaging and targeted therapy has shown promise in preclinical models of several solid tumor types -FEAU . To. ToTNF vn = 25) with varying tumor grades was quantitatively analyzed. Intensity of staining was denoted as strong (3+), moderate (2+) and weak (1+)  and low  staining intensities  to assess expression of the target integrin subunit αv in human gliomas and to support potential clinical applications of AAVP constructs. A cohort of human malignant gliomas (1+) Fig. . In ~28%ies Fig. . Locatioies Fig. . Most sties Fig. , arrows,TNF or HSVtk plus GCV as we design further translational studies. The cytokine TNF, for example, is an effective antitumor cytotoxic and may be used as a single-agent in repeat dose studies, greatly lessening the complexity of a treatment scheme. Imaging studies are routinely done to evaluate tumor response serially over time in glioblastoma patients [TNF, but not gene expression itself. Alternatively, HSVtk provides an opportunity to evaluate molecular transgene expression serially with a radiolabeled probe. The combination of targeted delivery of an HSVtk transgene, molecular-genetic imaging with a radiolabeled probe to noninvasively evaluate gene expression, and GCV suicide gene therapy might ultimately lead to a complex, yet elegant clinical trial design that could be tailored to individual patients who would be monitored over time to non-invasively monitor HSVtk gene expression. Once maximum expression is reached, GCV dosing could begin and both the transgene and tumor monitored over time. While persuasive, this may be a harder trial design to evaluate in patients, with multiple moving parts to consider in a clinical setting. Because gliomas are routinely imaged through MRI, targeted AAVP-TNF vectors would seem to have a straightforward clinical path. This is one reason why RGD4C-AAVP-TNF was chosen for translational application (phase zero to phase II) in a large cohort of pet dogs with native tumors [To ascertain the translational potential of the RGD4C ligand motif, we analyzed the efficacy of two AAVP constructs in an orthotopic model of glioblastoma. Gratifyingly, both the cytotoxic and theranostic versions of targeted AAVP showed antitumor effects with damage to the tumor-associated vasculature and evidence of apoptotic cells. However, one must consider pros and cons of targeted delivery of patients , so tumoe tumors  and it iThere are numerous gene therapy approaches being pursued in gliomas, recently reviewed by Caffery et al. . ClinicaMany studies succumb to a few commonly identified roadblocks to gene therapy against glioblastomas. Notably, poor transduction efficiency is a frequent occurrence, and several reasons have been postulated for inefficient cell transduction, including poor penetrance after intratumoral injections and lack of a viral receptor for viral vector gene therapy approaches . We atteEarly stage clinical trials utilizing either HSVtk or TNF in various viral constructs have been reported, which is encouraging for our ongoing translational efforts. Many of these trials have demonstrated both safety and some efficacy in patients with malignant glioma . The comFurthermore, although reproducibly sufficient gene expression has been found in several studies utilizing the AAVP gene delivery vector, transduction efficiency is still notably poor. One could speculate that this is not a fatal flaw in our case: both TNF and the combination of HSVtk plus GCV work via a bystander effect, maximizing the effect of even low transduction efficiency. In the case of TNF, we must be careful to avoid tipping the delicate balance of TNF signaling from apoptotic to proliferative due to intrinsic factors in the tumor microenvironment . In this


Reactive oxygen species (ROS) are important secondary metabolites that play major roles in signaling pathways, with their levels often used as analytical tools to investigate various cellular scenarios. They potentially damage genetic material and facilitate tumorigenesis by inhibiting certain tumor suppressors. In diabetic conditions, substantial levels of ROS stimulate oxidative stress through specialized precursors and enzymatic activity, while minimum levels are required for proper wound healing. Photobiomodulation (PBM) uses light to stimulate cellular mechanisms and facilitate the removal of oxidative stress. Photodynamic therapy (PDT) generates ROS to induce selective tumor destruction. The regulatory roles of PBM via crosstalk between ROS and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) are substantial for the appropriate management of various conditions. Reactive oxygen species (ROS) are formed by the fractional reduction of molecular oxygen and include, but are not limited to, superoxide anions, hydrogen peroxide, and hydroxyl radicals, all obtained from sequential oxidation–reduction processes involving nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, lipoxygenases, or cyclooxygenases . UnusualROS plays a pivotal role in the initial stages of wound healing by killing invading bacteria and other microorganisms. However, under chronic conditions, increased production of free radicals was observed, thereby inhibiting the proliferation and migration of key cell types and leading to delayed wound healing ,9. The rEven though PBM is well-known for its cell-stimulating properties both in vitro and in vivo, clinical studies have been very mixed, and some contradicted non-clinical studies ,13,14,15ROS are oxygen intermediates with unpaired electrons; both superoxide and hydroxyl radicals are highly unstable oxygen radicals . ExperimMitochondria and NADPH promote endogenous ROS formation in cancer and reports have shown crosstalk between these two producers . The mitOxidative damage is linked to all phases of cancer, during which ROS act throughout its progression. Radicals generated in the mitochondria damage genetic material and produce mutations that initiate tumor progression due to the imbalance between ROS generation and antioxidant defence mechanisms . Redox hGlucose metabolism is the mechanism behind the generation of oxidative stress. In a transition metal-dependent reaction, the enediol form of glucose is oxidized to enediol anion radicals, then converted to more reactive superoxide anions and ketoaldehydes. Through a dismutation reaction, superoxide anions are converted into hydrogen peroxides, which are precursors of highly reactive hydroxyl radicals. Under diabetic conditions, if these hydrogen peroxides are not degraded by glutathione peroxidase (GPx) or catalase (CAT), then the production of reactive hydroxyl radicals occurs in the presence of transition metals . SuperoxAmadori products and advanced glycation end-products (AGEs) are emerging as major precursors of free radicals in diabetes. AGEs may fluoresce, produce ROS, and bind to specific cell surface receptors . These gThe main processes involved in regulated wound healing are migration, adhesion, proliferation, neovascularization, remodeling, and apoptosis. Imbalance between antioxidants and free radicals with elevated ROS formation is often observed, followed by the induction of apoptosis. In chronic wounds, elevated free radicals coupled with reduced antioxidants results in the stimulation of pro-apoptotic transcription factors, caspase-3, and oxidative stress, leading to cell death and further delays in the healing process. The development of chronic impaired wound healing involves certain cell signaling reactions . FibroblUnder two different conditions, light is used to either attenuate oxidative stress (as in the case of PBM) or generate excessive amounts of ROS (as in the case of PDT). PBM involves the use of non-ionizing radiation or light specifically in the visible and near-infrared regions of the electromagnetic spectrum for therapeutic applications and stimulation of various cellular mechanisms. When there is an imbalance between antioxidants and free radicals, PBM facilitates the displacement of free radicals and the reduction of the oxidative stress load on the organism . The sucPDT is a minimally invasive treatment that is becoming commonly accepted as a potential therapeutic option for localized cancers . PDT depActivation of NF-кB depends on various parameters, like the cell compartment and dimer confinement. Cytoplasmic NF-кB dimers are maintained in a stationary and inactive state through their association with IкB (inhibitor of κB) proteins. When unconfined, NF-кB dimers translocate to nuclear regions and bind to targeted DNA sequences, promoting gene expression . Other dActivation of NF-κB can be observed by direct and indirect glutathionylation of NF-κB and I-κB, respectively. The p50 of cysteine-62 is sensitive to oxidation, which inhibits the translocation of NF-κB into the nucleus and prevents DNA binding . HoweverThe archetypical IкBα protein promptly degrades during the canonical activation route, causing the release of multiple NF-кB dimers. IкBα principally targets the p65:p50 heterodimer to form a IкBα: p65:p50 complex, which has translocating capabilities and masks the p65 nuclear localization signal. DNA binding is prevented by the IкBa nuclear export signal, which results in steady-state cytoplasmic localization of NF-кB dimers . The degHydrogen peroxide inhibits IKK activation by directly affecting IKKβ through its cysteine inhibitory ability in catalytic domains of tyrosine phosphatases. The interactions of ROS with cysteine residues are essential in order to affect the NF-кB pathways ,75. TheyMoreover, ROS appear as vital bridging factors that mediate the crosstalk between JNK and NF-кB signaling pathways and interconnect ROS-mediated NF-кB and cell death, with the ability to inhibit JNK activation . FurtherNF-кB is involved in various stages of oncogenesis and controls the expression of certain tumor-related genes. During initiation in pre-malignant cells, stimulated NF-кB promotes the expression of cytokines and chemokines, leading to the recruitment and activation of immune cells, which produce more chemokines and growth factors . They acNF-кB is involved in tumorigenesis by upregulating the anti-apoptotic pathway and tumor cell survival. The canonical pathway of NF-кB is known to activate the transcription of a group of anti-apoptotic proteins, which is further subdivided into two categories, namely, inhibitors of apoptotic proteins and the Bcl-2 family members . In addiInsulin-dependent (type 1) and insulin-independent (type 2) diabetes are the two major variations of diabetes seen among humans. In type 1 diabetes, beta pancreatic cells are prone to autoimmune attack by cytokines, such as interferon and IL-1, and NF-кB is mostly inactive in resting cells. However, IL-1 can activate the translocation of NF-кB to the nucleus . NF-кB rType 2 diabetes is mainly characterized by insulin resistance. NF-кB is one of the chief components in the insulin resistance observed in type 2 diabetes. The anti-inflammatory agent aspirin prevents the degradation of NF-кB inhibitor (IB), whose expression in the liver attenuates the expression of NF-кB dependent genes, but also reduced the likelihood of type 2 diabetes development. Similarly, inflammation-induced hyperglycemia can be inhibited using specific inhibitors of interleukin-1 signaling . Hence, NF-кB is essential for inflammatory and phagocytic cell migration as it strictly regulates genes involved in cell proliferation (granulocyte colony-stimulating factor and macrophage colony-stimulating factor), transformation, and survival. In wound healing, the classical NF-кB pathway is activated as an innate immune reaction and, as a result, activation of NF-кB is more important in the protection of cells from infections or microbes . The effUnusually high levels of ROS can allegedly be used for cancer diagnosis, varying according to tumor type, and are potent signaling molecules in cancer scenarios, thereby leading to nuclear damage, genetic instability, and tumorigenesis. The regulation of redox transcription factors is dependent on the generation of ROS levels. NF-кB was the first redox-regulated transcription factor to be discovered. It is a protein complex with multiple functions in immune, inflammation, cell growth, and survival responses. ROS have NF-кB stimulatory effects in the cytoplasm and inhibitory effects in the nucleus. Therefore, ROS have the ability to both activate and suppress NF-кB signaling pathways. PBM promotes photon availability and absorption by chromophores in the catalytic center of mitochondrial cytochrome c oxidase the and subsequent reduction of molecular oxygen. This disturbs the mitochondrial membrane potential and changes the fission–fusion homeostasis in the mitochondrial network, leading to increased levels of intermediates such as ATP, cAMP, and ROS. PBM-mediated ROS interact with certain family proteins that enclose highly reactive cysteine (Cys) residues and after the oxidation of Cys residues. The activity of NF-кB is regulated by direct activation or after disassembling from IkB, an inhibitor, then the released NF-кB translocate to the nucleus and promotes gene transcription. The regulation of crosstalk between ROS and NF-кB appears to be a key factor for better management of many conditions. PBM facilitates the reduction of oxidative stress and activation of mechanisms that upregulate or downregulate NF-кB, which are critical for the promotion of anti-apoptotic responses and tumor progression, insulin dependence or resistance in diabetes, cell proliferation and migration in wounded sites, or cell survival by ROS attenuation.


Targeted drug delivery system improves the efficiency and safety of the therapeutic agents by managing the pharmacokinetics and pharmacodynamics of drugs. Currently, numerous drug carrier systems have been developed with different sizes, architectures and characteristics surface properties. Among different systems, macromolecules have a wide range of applications in targeted drug delivery system. The optimal drug loading potential, smooth drug releasing ability and biocompatibility are the distinguishing features that ensure the drugs delivery ability of macromolecules. This review briefly introduces some of the most commonly studied macromolecules which have been recommended as drugs delivery vehicles. Targeted drug delivery system (TDDS) is a distinctive technique where the drug is administered to the desired location instead of the whole body or organ . The selThe applications of TDDS are observed in various clinical conditions, including various types of cancer treatments. Chemotherapy has become an integral component of for most cancers treatment. Despite the last 30 years of effort on oncology drug discovery, conventional chemotherapeutic agents still exhibit poor specificity in reaching tumour tissues and are often restricted by dose-limiting toxicity. Concurrent and adjuvant/neo-adjuvant chemotherapies are usually practised along with the radiotherapy and surgery .The concept of TDD dates back to 1906 when Paul Ehrlich (German Nobel laureate) proposed ‘magic bullet’ . HoweverLiposomes are among the commonly studied drug delivery systems . The worThe significant physical and chemical properties of liposomes are due to its main constituent, phospholipids . The phoFunctionalization of liposomes could be attained through various techniques. Among these, adsorption method is an easily approachable method where liposomal dispersion is incubated with the solution of targeting agent. However, the rapid desorption rate during storage and in vivo system make it less superior . An alteTo ensure the encapsulation of hydrophobic drugs into the inner hydrophilic core of liposome, new approaches have been developed that allow loading of hydrophobic substances in the inner aqueous part of liposome. Okamato et al., reported albumin-encapsulated liposome for the encapsulation of hydrophobic molecules (drugs). The modification was based on the assumption that attachment to albumin enhances aqueous solubility of hydrophobic agents. The size of bovine serum albumin(BSA)-liposomes can be regulated so as to target inflammatory sites and tumors via the EPR effect .Various stimulants are used for the smooth detachment of therapeutic agents from the liposomes. The release of drug in the intended site is subjective to the membrane composition of the liposome and the nature of drugs attached to the liposomes . The pH Polymer micelles (PM) are promising vehicles that solubilise as well as carry/deliver poorly water-soluble therapeutic agents. Micelles are typically made up of 50–200 monomers. The diameter of PM ranges from 10–100 nm . The sizin vivo system encourage its accumulation within the tumour cells arene is an example of host-guest chemistry. In silico study of imatinib-calixarene complexes by Galindo-Mrillo suggested the controlled delivery of the drug (imatinib) into the cancer cells [Calix[n]arenes are cyclic oligomers that are known as the 3o twenty . A calixo twenty . The Cuper cells . However2-hybridized carbon atoms are organised hexagonaly (µm [Carbon nanotubes (CNTs) were discovered in 1991 by Prof. Iijima . These nxagonaly . Based onaly  are cyclic oligosaccharides linked by α-1,4 glycosidic bonds which havetorus-like molecular shape with hydrophilic outer face and a lipophilic central cavity . Most co(EPR) effect. More so, Polymer-drug conjugates prolong the drug circulation times in the body, and protect the drug from premature degradation. Rings dorf (1975) introduced this concept and a few years later, Duncan & Kopecek designed the first targeted polymer-anticancer conjugates [N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers [Polymer-drug conjugates are among the commonly studied therapeutic carriers where a drug is covalently attached to the polymer, either directly or through a linker . There are three main components of a polymer-drug conjugate, a soluble polymer backbone, a biodegradable linker and covalently attached therapeutic agent(s) . A linkenjugates . The arepolymers .The aqueous solubility, biocompatibility (the coexistence with living tissues without harm) and commercial availability are the characteristic features of PEG . PEG pos8.by cis-trans interchange of the azo part by an external stimulus (light or temperature). For example, Tong et al.synthesised amphiphilic diblock copolymer made of azo benzene polymethacrylate (PAzoMA) and poly(acrylic acid)(PAA) [trans-cis photoisomerization was observed in the azo part by changing the UV/Vis light irradiation, that resulted in change in the morphology of polymer. Saint-Cricq et al. designed azo containing degradable nanoparticles for targeted drug delivery [Compounds containing at least one – N = N- functional group are called azo compounds. They have been reported as drug carriers by encapsulating drug molecules or by prodrug approach. In prodrug pathway, the therapeutic agents are conjugated via azo linkage and are subsequently released by the action of azo reductase enzyme. The azo reductase sensitive system is useful in colon-targeted drug delivery for the treatment of related diseases, such as colorectal cancer, inflammatory bowel disease and amoebiases . Variousid)(PAA) . A reverdelivery . The nanTargeted drug delivery system decreases the adverse effects of chemotherapy. Several drug carriers with different sizes, architectures and characteristics surface properties have been suggested for targeted drug delivery. Among these, macromolecules have been declared as potential candidates due to their optimal drug loading potential and smooth drug releasing ability. Drugs molecules can easily be attached to conjugated polymers, polymeric micelles, liposomes, dendrimers, polymeric nanoparticles due to their characteristics structural features. Stimuli-responsive azo-based compounds are useful in colon cancer and related diseases. Further consideration and interdisciplinary research might help to introduce a safer way of treatment, especially in the challenging field of cancer chemotherapy.


Atomic force microscopy (AFM) is a pioneer imaging technique commonly employed by biological researchers in detection of the properties of biological membranes over the last decade. The AFM findings distinguish its applicability from the conventional methods, such as: confocal, multi-photons, electron microscopy, etc. as well as from the mechanical methods . With its high resolution (below 10 nm), AFM has emerged as a powerful tool in obtaining the nanostructural details and biomechanical properties of heart tissue. The composition of extracellular matrix is essential for heart compliance and its mechanical function. Here, we illustrate the surface morphology, its structural assembling and the mechanical properties of a myocardial infarction scar section aquired via AFM, in dry conditions. The cross section through the mature myocardial scar of mice after myocardial infarction shows that the embedded fibrils into the tissue matrix of a mature scar overlap at some sites, and form network-like structures. The nano-fibrils surface shows defined structural periodicity. A cross-section along the axial fibrilar direction gives an average D-periodic banding pattern of approximately 50,3 nm . As future perspective, yet uncovered morphological and mechanical investigations, correlated with functional studies, open a new window for understanding pathological mechanisms. Changes in the two competitive physicochemical parameters of collagen formation and desintegration rates lead to compositional modification of extracellular matrix, which in turn modifies the heart tissue compliance, resulting in a change in heart function4. Of a great interest are the nano-assemblies of structural subdomains forming collagen fibrils, which lie between molecular and fibrillar levels. The substructural information relates to the overall mechanical properties of fibrils, by preventing or developing cracks. These cracks may predominantly propagate in the border zone (delineated as the region between scar and remote zones), when undergoing post-myocardial infarction (data not shown). The surface of the fibril is the “mirror” of all interface interactions with adjacent fibrils or other molecules. State of the art morphological techniques, such as AFM, are providing valuable information on fine structural and morphological details, such as molecular heterogeneity5. Microscopically, collagenous structures appear as elongated micro-fibrils packed into fibrillar collagen types. Their periodic banding patterns depend on collagen composition and the synthesized sites6.Atomic force microscopy (AFM) uses a scanning probe microscope based on the interaction of a sharp tip and the underlying atoms of the investigated surface. AFM is a unique research tool to investigate surface properties  in three-dimension. The AFM findings will open a wide avenue mostly in the cardiovascular field, where heart function need to be associated with structural changes and functionality. AFM has emerged as a powerful tool to obtain the nanostructural details and mechanical properties of biological samples, including of biomolecules and cells. Remodeling after myocardial infarction starts with a massive extracellular matrix production and deposition as a vectorial mechanism towards fibrosis formation. The cascade of infarction events is reflected in tissue function loss, where the contractile elements start the vicious cycle of mechanical inefficiency and eventual heart failure. Collagen is the main component of the extracellular matrix, providing strength to various structures. The scar tissue formed after an ischemic event is involved in preserving the mechanical structure of the heart, sustaining the pump function of the heart muscle. Scar formation is a dynamic process, starting early after myocardial infarction with synthesis of a fibrin-based matrix. Later, the myofibrobalsts are modulated by numerous factors to synthesize a dense collagen-based scar. Our knowledge with respect to biochemical composition, structure and functionality in the scar tissue at the molecular level is just at the beginning. In myocardial infarction, fibril-forming collagen I and III stabilized the scar, while non-fibrilar collagens IV and VI have an impact on myofibroblasts differentiation and organization of the extracellular matrix network. Furthermore, extracellular matrix proteins, including fibronectin, elastin, laminin, biglycan and other components are required for proper healing of injured myocardial tissueFigure 1, (A) the cross section through the mature myocardial scar of mouse is visualized in a dry state by peak force tapping quantitative nano-mechanics AFM. The embedded fibrils into the tissue matrix of a mature scar overlap at some sites (yellow circles), and form network-like structures (yellow head arrow). Few sites represent the anchoring points (blue head arrows) for some nano-size fibrils, while some fibrils seem to be completely detached (green head arrows). The nano-fibril’s surface shows defined structural periodicity. The nano-fibril’s contour has a well-defined topological profile, while some bundles of fibrils have a faint color. (B) The identified periodical structures recorded along the axial direction are depicted with an alternating bright and dark color gradient in the adhesion map. The alternating pattern appears with a spatial distribution of approximately 50 nm. The bright color assigns a low adhesion profile, while the soft nano-segments are illustrated in dark. (C) Topographic analysis perpendicular to the longitudinal axis of fibrils suggests a mean fibril height of 20.3 nm (± 4.2 nm std.), whereas a cross-section along the axial fibrilar direction gives an average D-periodic banding pattern of approximately 50,3 nm .In the Our understanding of the regulatory mechanism involved for fine-tunning the extracellular matrix formation and degradation is fundamental for the development of agents that may serve or prevent excess collagen deposition in cardiovascular diseases.


Traumatic spinal cord injury results in severe and irreversible loss of function. The injury triggers a complex cascade of inflammatory and pathological processes, culminating in formation of a scar. While traditionally referred to as a glial scar, the spinal injury scar in fact comprises multiple cellular and extracellular components. This multidimensional nature should be considered when aiming to understand the role of scarring in limiting tissue repair and recovery. In this Review we discuss recent advances in understanding the composition and phenotypic characteristics of the spinal injury scar, the oversimplification of defining the scar in binary terms as good or bad, and the development of therapeutic approaches to target scar components to enable improved functional outcome after spinal cord injury. The scar formation that occurs following spinal cord injury has properties that are distinct to scars seen in other areas of the CNS, and in other tissues. Here the authors discuss the components of the spinal cord injury scar and how it can have both detrimental and positive roles in relation to recovery. Following traumatic spinal cord injury, death of spinal neurons at the injury level leads to paralysis of denervated musculature and the disruption of long spinal tracts leads to loss of sensation and motor control—injured descending axonal projections can no longer innervate motor neuron pools below the injury level, and injured ascending axonal projections can no longer provide appropriate transmission of sensory information to the brain. This results in the dysregulation of multiple organ systems throughout the body and a devastating loss of function2. Despite recent progress in developing experimental therapeutics aimed at enhancing tissue repair and neuroplasticity, there are still no effective pathology-modifying or regenerative treatments available to spinal injured individuals4. Following diagnosis and acute medical interventions to stabilize clinical status, the outcome is largely determined by the management of resultant symptoms, and rehabilitation to maximize residual neural function2.It is estimated that more than 27 million people worldwide are living with long-term disability following a spinal cord injury5. Experimental efforts to unlock regeneration potential at the level of the cell body of the neuron have focussed on growth signalling pathways, individual regeneration-associated genes, and transcriptional and epigenetic networks9. Regenerative strategies have also aimed to increase synthesis and transport of materials required for growth, and to modulate axonal cytoskeletal dynamics to promote elongation or branching12.The lack of repair following spinal cord injury is due to both cell intrinsic factors and the extrinsic injury environment. Neurons of the adult mammalian central nervous system (CNS) have low intrinsic regenerative ability due to a lack of growth driving signals, and suboptimal availability or arrangement of subcellular machinery to enable growth cone reformation and axonal elongationThe injury microenvironment also plays a key role in limiting functional repair after spinal cord injury, an important component of which is the formation of a scar. As a healing response, the scar acts to spatially contain and isolate damage. However, reactive injury processes fail to restore spinal tissue architecture and composition, pathology continues to propagate and the tissue within and around the scar remains dysfunctional. Moreover, within the scar there are extracellular factors which themselves actively inhibit restoration of function. These act acutely and chronically to prohibit compensatory changes in neurons which, perhaps if overcome, could transform the scar into a more effective repair process which both isolates damage and generates an environment in which injury could be surmountable.Here we review recent literature regarding the composition and role of the spinal injury scar, including processes leading to scar formation and maintenance, the cellular and extracellular components of the scar, and how these interact with other mediators of tissue pathology. We discuss the complexities of the scar and its seemingly opposing roles, often classified in an overly simplistic manner as good or bad, and finally we discuss the potential for therapeutic targeting of the scar to achieve functional repair of the injured spinal cord.13. However, the process of tissue scarring in the CNS is more complex than for many other tissues, and is associated with chronic non-resolving pathology.Injury to any tissue results in a healing response, the purpose of which is firstly to curtail damage and restore homoeostasis and then, where possible, to restore tissue and organ function. Inflammation, tissue reformation (involving cell proliferation and/or migration) and tissue remodelling are conserved repair processes, although their success in restoring function varies across different tissues. A scar consists of the cells and extracellular matrix (ECM) formed as the result of attempted wound repair. In many organs, the formation of a scar is associated with a resolution phase and restoration of key functions of the tissue. The healed tissue may not directly recapitulate the pre-injury state but it regains some ability to execute its original function14 and stroke15, as well in neurodegenerative disorders such as Alzheimer’s Disease16, and disorders with a predominantly demyelinating and inflammatory pathology such as multiple sclerosis18. However, despite the occurrence of reactive tissue changes and scarring in several other CNS pathologies, spinal cord injury represents a particularly striking example where wound repair is inefficient and injury-induced pathological changes are insurmountable.Disease and injury to the CNS is almost always accompanied by some degree of reactive gliosis, inflammation and scarring. Scar tissue and associated deposition of ECM molecules such as chondroitin sulfate proteoglycans  has been reported in humans and experimental animal models following traumatic brain injury19. There are also differences in the levels of neuroinflammation and astrocyte activation after brain and spinal cord trauma, with increased expression of inflammatory cytokines and damage-exacerbating leukocytes22 and more abundant and widespread astrocytosis in spinal cord injuries compared to brain injuries20. There are also differences in ECM composition and distribution between brain and spinal cord pathologies25. In this Review we specifically focus on scarring following spinal cord injury, and discuss the dynamic cellular and extracellular interactions that culminate in a hostile scar environment with limited capacity for repair.There are several likely contributing factors to these regional and injury-specific differences in CNS scarring. Cell types involved in scarring in different CNS regions are phenotypically different28. The term glial scar has historically been used to describe the astrocyte border component of the scar31, although some investigators use the term more widely to reflect the entire lesion including both glial and non-neural components32. Other distinctions have been made between the glial scar and the fibrotic scar33, and specific components of the lesion  have recently been referred to as lesion-related tissue compartments31. While these distinctions are valid, and scar components undoubtedly become spatially compartmentalized in a chronic scar, these components are nonetheless interlinked, and evidence suggests that there may be temporal dependence and bi-directional cross talk between them. Furthermore, the scar should not be considered as an isolated component of spinal injury pathology since it is shaped by processes of inflammation and tissue and matrix remodelling. Here we use the term spinal injury scar, which encompasses both cellular and extracellular components across the lesion core, lesion border and surrounding penumbra . Tissue deformation transmits shearing and compressive forces on axons and blood vessels and initiates a cascade of pathological processes. Below, we discuss these processes, focusing on changes occurring immediately after the initial spinal cord trauma up to one day post injury. These acute post-injury events signal the beginning of the injury cascade, which culminates in chronic pathology and scarring , microglia and astrocytes express a heterogeneous mix of P2 receptor subtypes, and respond reactively to increasing levels of ATP following trauma41.Vascular trauma leads to haemorrhage, and accumulating blood sera increases tissue colloid osmotic pressure, causing local oedema and swelling42. ATP release, dramatic loss of cellular and extracellular ionic homeostasis and excessive calcium levels results in the activation of calpains, phospholipase A2 and lipoxogenase. This is followed by the generation of bioactive lipid mediators and free radicals43. Progressive oxidation of fatty acids in cell membranes and myelin (lipid peroxidation) occurs. Furthermore there is feed-forward propagation of the injury and cell reactivity, as bioactive mediators potentiate ATP-mediated calcium increases in glia40 and additional blood vessel endothelial cell damage results in an expanding zone of haemorrhagic necrosis44.Subsequently, tissue reperfusion induces further oxidative stress, glutamate release and death of neighbouring neurons and glia via excitotoxicity45 is associated with reactive changes in resident glia and represents a potent inflammatory stimulus. Platelets themselves are an abundant source of inflammatory peptides and protein mediators and release cytokines, chemokines and eicosanoids which readily communicate with resident spinal cord cells and non-resident leukocytes. These signals cause rapid neutrophil infiltration within an hour (peaking within 24 h), which secrete MMP9, a type IV collagenase which acts on basement membranes to further permeabilise the blood brain barrier34. Furthermore, cellular and extracellular factors  constitute host-derived danger signals46. Damage associate molecular patterns (DAMPs) are sterile inflammatory stimuli  which activate prototypical pathogen recognition receptors  to induce secretion of proinflammatory cytokines and chemokines from both neurons and glia, compounding reactive gliosis and acting to recruit circulating immune responders50.Concomitantly, haemostasis is the first stage of wound healing: endothelial cell trauma results in platelet adhesion and activation, the coagulation cascade, and thrombin-mediated conversion of fibrinogen to fibrin, to form a clot. The onset of hemostasisNon-resolving pathology results in incomplete tissue repair and formation of a scar. Each cell type that contributes to this pathology, and its respective phenotype, is inherently linked to the environment it finds itself in. This environment constitutes a milieu of other resident and non-resident cells, the signals they transmit and the biochemical and biophysical properties of the extracellular environment in which they reside. Below, we discuss cellular and extracellular changes occurring in the sub-acute period, from days to several weeks after the initial spinal cord trauma, and their contribution to the spinal injury scar. Cellular and extracellular components of the spinal injury scar in the acute and chronic phases of spinal cord injury are depicted in Fig. 51, and ranges from temporary changes in gene expression and cell morphology to significant hypertrophy, spatial rearrangement and proliferation (collectively termed astrogliosis)53. Additionally, diverse astrocyte subsets and phenotypes may exist following spinal cord injury  and the organization of astrocytes into a barrier-like structure57.Astrocytes become reactive following spinal cord injury. The degree of reactivity is influenced by a number of cell-surface receptors for DAMPS and proinflammatory cytokines and chemokines59 and leucine zipper-bearing kinase  expression56. Ependymal cell-derived astrocyte-like progeny contribute to this population of astrocytes in certain spinal injury models60, athough evidence suggests this contribution is minor following contusive injuries61. Tight linking of astrocytes at the injury borders is associated with reformation of the glia limitans and the containment of immune cells and fibroblast-like cells within the injury epicenter, via ephrin-mediated cellular adhesion63. Thus, a population of reactive astrocytes act to spatially isolate damage and fibrosis from spared tissue.This barrier is reinforced by proliferation and organization of a local astrocyte population at the injury border, thought to be mediated via STAT-3 dependent signalling64. Though astrocytes alone are not responsible for the formation of a scar, they are major cellular players activated and maintained during post-injury pathology, inflammation and tissue and matrix remodelling. Alongside other cells and extracellular factors, astrocytes shape the scar cellular and extracellular milieu sub-acutely and chronically.An overlapping population of astrocytes are further associated with the chronic maintenance of this structure. These are referred to as scar forming astrocytesFibroblasts are ubiquitous in peripheral connective tissues and organs and are the principal generators of stroma, including the ECM. By contrast, under normal conditions within the CNS, fibroblast-like cells are mostly associated with the vasculature, and contribute only to the basal laminae. However, injury to the spinal cord induces a significant fibroblast response which produces matrix components. These matrix components may inhibit neural regeneration directly, and promote prolonged tissue remodelling via interaction with inflammatory cells (detailed below). These stromal elements become spatially compartmentalized by surrounding reactive astrocytes to form the fibrotic core of the spinal injury scar.33 and can derive from perivascular cells in rats65 and mice63 following contusion injury. There is some evidence that fibroblast-like stromal cells derive from pericytes; the PDGFRβ+ Glast+ perivascular cell population, termed type A pericytes, proliferate in response to injury and contribute to fibrotic scarring66. Preventing Glast1+ cell proliferation leads to failure of wound sealing, exacerbated lesion volume and decreased matrix deposition66, whereas moderate reduction of pericyte-derived fibrosis was found to reduce scar pathology and confer functional recovery67. However, whether this is a truly separate population or whether it overlaps with known cell types (such as Glast+ astrocytes in the glia limitans) is unclear.Fibroblasts proliferate from meningeal cells if the dura is compromised68. However, NG2 is also a marker of OPCs, Schwann cells and macrophages following spinal cord injury69. Thus, there is some ambiguity as to the response of pericytes following injury, although collectively there is evidence that fibroblast-like cells are derived from a perivascular PDGFRβ+ origin70.An increase in type-1 pericytes (distinct from those described above) has also been described, following a non-contusive dorsal funiculus lesion using a nestin-GFP/NG2-DsRed transgenic mouse line72. Two main contributions of OPCs within the spinal injury scar environment have been described, with seemingly opposing roles. OPCs contribute to remyelination, either by oligodendrogenesis or through differentiation into remyelinating Schwann cells75, and also hypertrophy and increase expression of NG2, a proteoglycan thought to mediate entrapment of neurons76. However, the roles of OPCs in the spinal injury scar have been obscured experimentally by overlap of markers with other cells. Alongside NG2, PDGFRα, two ganglioside antigens, and a cyclic nucleotide phosphodiesterase , OPCs also express the traditional astrocyte marker GFAP, and may differentiate into a de novo population of astrocytes in the scar77. Additionally, both PDGFRα and NG2 are also thought to be expressed by at least one type of perivascular/pericyte-type cell71. The use of fate mapping transgenic mouse lines, such as those expressing Cre recombinase under control of the PDGFRα promoter/enhancers78, alongside inclusion/exclusion of specific markers, will further define their role and contribution to scarring after spinal cord injury.NG2+ OPCs become reactive after spinal cord injury, have a significant proliferative capacity and are spatially intermingled with other reactive glia at the injury border+, CD45high and microglia CD11b+, CD45low) or chimeric models. Specific, or enriched, markers for microglia have now been discovered, including transmembrane protein 119 (Tmem119)79, P2ry12 and Fc receptor-like S (FCRLS)80. Tracking resident microglia via a genetic strategy has shown that spared microglia proliferate and repopulate the lesion core alongside monocyte-derived macrophages38 and recent availability of Tmem119 reporter mice82 will further elucidate the role of microglia in spinal injury scarring.By 24 h after spinal cord injury, blood-derived monocytes are recruited into the lesion. Upon extravasation, DAMPs and the associated reactive and inflammatory environment shape their differentiation phenotype. Meanwhile, resident microglia retract cellular processes and become morphologically indistinguishable from infiltrated monocyte-derived macrophages. Until recently, microglia and macrophages were only distinguishable using relative gene expression of CD45  to pro-repair . Following spinal cord injury, there is initially a mixed M1/M2 response86. Other adaptive immune system components are recruited by 7 days, and contribute to non-resolving trauma-induced autoimmunity87. Leukocytes present DAMP-derived antigens (such as MBP) to T and B-cells. B cells, in turn, may present antigens to T-cells, triggering their expansion. Furthermore, B-cells differentiate into plasma cells synthesizing auto-antibodies, further fuelling a feed-forward immune response88.The adaptive immune system also plays a role. After spinal cord injury, the recruitment of γδT cells, and production of proinflammatory IFNγ occurs within 24 h following injury89. Macrophages maintain 45% of peak activation months after injury90 and their phenotype does not undergo the switch from pro-inflammatory to pro-repair associated with the next phases of wound-healing in other organ tissues91. The early arginase1+ (M2-like) differentiating infiltrating population is not maintained83 and the spectrum of innate immune cell activation phenotype is predominately M1 polarized. Adaptive immunity is non-resolving, whereby lymphocytes remain indefinitely in the spinal injury scar.Unlike conditions in which successful wound healing occurs, there is no effective resolution to cellular recruitment and inflammation after spinal cord injury. Monocyte or microglia-derived macrophages remain in the injured spinal cord indefinitely92 or NFKB93 increases monocyte infiltration to the lesion epicentre. In addition, resident and infiltrated microglia/macrophages express a number of receptors for proinflammatory chemokines and cytokines released by reactive astrocytes , contributing to a cell signalling environment which potentiates M1 polarization. In turn, astrocytes express receptors for a number of inflammatory mediators released by immune cells , inducing extensive astrocyte reactivity and astrogliosis94. Thus, there is an intimate link between astrocytes and resident and infiltrating immune cells during formation of the spinal injury scar. Astrocyte-fibroblast interactions have been shown to spatially compartmentalize the fibrotic core62 and recent evidence suggests that microglia may provide an additional interface between these cells, which the authors term the microglial scar38.The response of astrocytes, OPCs, microglia and infiltrating innate and adaptive immune cells is continually influenced by one another and the tissue environment  leads to a trophic A2 polarization state. By contrast, activated microglia from models of neurodegenerative and neuroinflammatory diseases and traumatic optic nerve crush injury release factors such as IL1α, TNF and complement component subunit 1q (C1q) which induce a neurotoxic state in A1 astrocytesNes, Ctnnb1, Axin2, Plaur, Mmp2, and Mmp13 whereas scar-forming astrocytes selectively upregulated Cdh2, Sox9, Xylt1, Chst11, Csgalnact1, Acan, Pcan and Slit254. Furthermore, using these genes as population markers, FACs isolated nestin-GFP+ reactive astrocytes were found to convert to a naïve phenotype following transplantation into uninjured tissue but become scar-forming when transplanted into injured tissue, an effect thought to be mediated by a N-cadherin-dependent interaction with type 1 collagen54. Thus, astrocytes are able to display phenotypic plasticity, and tissue environment is a crucial influence over cellular behaviour.Genetic profiling of reactive astrocytes and scar-forming astrocytes following spinal cord injury  has recently been reported. Reactive astrocytes were associated with selective upregulation of 108. Two weeks after a spinal crush injury RNA-seq revealed differential expression of over 6000 genes in astrocytes, changes described as congruent with prior transcriptomic analysis following ischemic stroke lesion108. However, whether the astrocytes sampled here represent A2-like trophic astrocytes has not been ascertained.The GFAP-RiboTag mouse can be used to perform high-throughput RNA sequencing on astrocytes following injury and probe the effect of particular genes in the astrocyte response54, the RNA-seq dataset only supports increased expression in one of these (Xylt1)108, whereas four of the six genes ascribed to reactive astrocytes54, are increased 108. Spatial differences in sample selection may contribute to these disparities. Techniques such as 3D intact-tissue RNA sequencing182 may overcome this problem in the future. Indeed, further characterization of spatio-temporal phenotypic diversity and plasticity would aid research into how astrocyte phenotype and scar progression may be modified by changes to matrix components or perturbation of the immune response. Such methods, alongside new purification techniques for in vitro analysis, will likely provide increasingly nuanced understanding of the relationship between astrocytes, microglia, non-resident immune cells and the tissue environment185.There are some discrepancies between gene expression findings using these different methodologies. For example, of the eight genes associated with scar-forming astrocytes isolated using laser capture microdissection101. Following injury, resident glia and stromal cells, which do not normally contribute parenchymal matrix, begin to contribute matrix components, and extracellular DAMPs are present in both sub-acute and chronic phases.The CNS ECM is rich in glycoproteins and proteoglycans. Hyaluronan forms a backbone for the attachment of tenascins and sulphated proteoglycans, stabilised by link proteins. This is arranged either diffusely in the interstitial space or more densely assembled around the cell soma of particular neuronal subtypes , or around axonal nodes of ranvier or synaptic boutons. These structures confer neural stability, localizing molecules such as CSPGs, which effectively restrict large-scale plasticity following a critical period in development50) and many of these play a role in neuroprotection or spontaneous repair and are not refractory to recovery. Fibrous matrix forms a seal or tissue bridge between retracting lesioned parenchyma. This is particularly apparent in injuries where spinal tissue is rendered non-continuous, for example, following hemisection or transection. Fibroblast-derived collagenous matrix is also a major component of the ECM33 (and in vivo ablation of fibroblasts compromises tissue integrity following injury)66. Basal laminae is restored via matrix deposition of collagen VI, nidogen, fibronectin and laminin, which are traditionally neuronal-growth permissive molecules. However, in the context of spinal cord injury, such ECM molecules are also implicated in pathological tissue remodelling or inflammation. For example, fibronectin, matrix glycoprotein tenascin C and hyaluronan fragments also act as endogenous TLR ligands and represent DAMPs48.A vast number of ECM molecules undergo differential regulation following spinal cord injury  upregulate expression of the ECM molecule periostin, which in turn upregulates TNFα expression from infiltrating monocyte-macrophages and leads to proliferation of PDGFRβ+ cells, type-I collagen deposition and fibrosis104, preventing axons from overcoming the spinal injury scar environment. Transmembrane receptor complexes are identified, converging on the canonical RhoA/ROCK signalling pathway, resulting in destabilization of the actin cytoskeleton and local arrest and collapse of growth cones105. Furthermore, CSPGs 113, NgR1 and NgR3114. Signalling pathways implicated have convergence with those of Nogo and other myelin inhibitors and include the Rho/ROCK pathway, activation of which is partly via PKC115 and EGFR116 and coupled to Akt/GSK-3 activation117. CSPGs are also thought to inactivate neural intergrins118 and localise upregulated inhibitory guidance molecules such as semaphorin 3A119. Thus, the injured spinal cord extracellular environment contains molecules which restrict neurite outgrowth and plasticity, and these are further upregulated and concentrated in the spinal injury scar and represent therapeutic targets120 (discussed below).In addition to an ongoing DAMP role for myelin debris, myelin-associated molecules confer extrinsic inhibition to neurons. These include Nogo A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp). Nogo-A is a potent inhibitor to neural plasticity and regeneration following spinal cord injurySPGs Box  are upreBox 2 Structure of chondroitin sulfate proteoglycans (CSPGs)50. Lecticans are the most abundant CSPGs in the spinal injury scar and also feature globular domains: the G1 N-terminal domain and G3 C-terminal domains are important in their interaction via link-protein with hyaluronan (the backbone glycoprotein of the CNS matrix) and also tenascin, thus they are involved in matrix crosslinking. Core PGs undergo post-translational modification in the endoplasmic reticulum and golgi, catalysed by a number of enzymes. At particular serine residues a tetrasaccharide linking region is formed by sequential addition of xylose by xylosyl transferase, two galactose molecules by β1,4-Galactosyltransferase-I then β1,3-Galactosyltransferase-II and a one GlcA residue via β1,3-glucuronyltransferase I to form the linker GlcAβ1–3 Galβ1–3 Galβ1–4 Xylβ1–O-Ser. The following addition of GalNac by a GalNac transferase I is crucial to initiate synthesis of the chondroitin sulfate backbone. If, at this point, N-acetylglucosamine (GlcNac) is added rather than GalNac, synthesis of the heparan sulfate backbone is initiated186. CS-GAG chain polymerisation is the process by which alternating residues of GalNac and GlcA are then added to the proteoglycan linker region by the alternating activity of a GlcA transferase II and a GalNac transferase II. There are six actual enzymes identified which confer this glycosyltransferase activity. Alongside CS-GalNac transferase I and II, combinations of enzyme complexes of chondroitin synthase 1, 2 and 3 and chondroitin polymerising factor (ChPF) mediate GAG polymerization.CSPGs are proteoglycans (PGs) consisting of a core protein with at least one covalently attached chondroitin sulfate glycosaminoglycan (CS-GAG) side chain (see figure). CSPG subtypes most commonly studied with respect to the inhibitory CNS environment include lecticans , the transmembrane protein NG2, phosphacan (transmembrane or soluble) and the small leucine-rich proteoglycans decorin and biglycan. There are also multiple less-well studied CSPGs revealed by proteomics analysis of scar matrix121. Cells are highly mechanosensitive and changes in the elastic properties of the environment alone can induce differentiation and migration122, and during development mechanical gradients guide axon pathfinding123. Astrocytes that are cultured on less compliant, stiff substrates anatomically resemble reactive astrocytes, displaying hypertrophy and elongation, with stiffness of CNS implants correlating with induction of reactive astrocytosis124. Integrin-mediated links between fibrillar type 1 collagen (known to be stiffer in other tissues) and astrocyte reactivity are emerging54, which may be influenced by mechanotransduction. Atomic force microscopy has been used to characterize the spatiotemporal elastic stiffness properties of spinal cord tissue over 1 to 3 weeks following dorsal column crush lesion125. At these early post-injury time points, tissue softened in areas corresponding to scarring and ECM deposition. This was somewhat surprising because scar tissue outside the CNS is typically stiffer than surrounding healthy tissue126, and was attributed to a lack of collagen-1 and loss of CNS myelin in these types of injuries 128, as well as the cellular composition of the scar . It will be important to further characterize these properties in more clinically relevant contusion-type injuries and in chronic injuries with established scar tissue, particularly given the increasing evidence of a role for collagen-1 in chronic contusive injuries129.Relatively little attention has been given to how the biomechanical environment of the injured spinal cord affects repair132. We propose that these two different viewpoints reflect different interpretations of data which is, in fact, largely in agreement. Both historical and newer findings support a long-established principle, that the spinal injury scar performs dual, and seemingly opposing, roles; to protect tissue, and to inhibit repair.There has been some recent debate in the field on whether the scar is good or bad in terms of recovery from injury30. An inhibitory role for the scar and scar-associated molecules has been well documented ever since (as discussed above and134). However, it has also long been acknowledged that the astrocytic scar has an important protective role in enabling the separation of healthy tissue from pathology following injury137. Thus, for decades the dual notions that the scar is associated with failed axonal re-connectivity (inhibitory) and also involved in a wound-healing response (protective), have existed.As previously introduced, the classical description of the spinal injury scar is one which considered the astrocyte-rich injury border alone, termed the glial scar. Early observations of dense glial reactivity at the site of CNS lesions led to the hypothesis that the astrocytic scar inhibits axon regeneration, perhaps by forming an impenetrable barrier to axonal extension138. Similarly, conditional ablation of proliferating (scar-forming) reactive astrocytes following injury increases edema, inflammation, oligodendrocyte death, tissue loss, demyelination and functional deficits140. Furthermore, deletion of SOCS3 or STAT3 in astrocytes results in lesion expansion, cell death and exacerbated functional outcome92. Thus, a number of studies have provided evidence for the importance of reactive astrocytes in preventing expansion of pathology into spared peri-lesional regions of the spinal cord. A recent extension of these studies utilized these deletion strategies to specifically assess the effects of reactive astrocytes on axonal regeneration108. The study revealed that spontaneous regrowth of damaged axons does not occur across a spinal crush injury following attenuation or ablation of scar forming astrocytes, despite boosting the regenerative state of these axons with a conditioning lesion and neurotrophin delivery108. This study claimed to reveal a new  role of the astrocytic scar as being pro-regenerative. Our interpretation of these findings, however, is that they are largely consistent with previous studies140. A lack of axon regeneration after ablating reactive scar astrocytes does not necessarily mean that the glial scar aids axon regeneration. An alternative interpretation is that the regenerative boost afforded by conditioning lesions and neurotrophin delivery is not sufficient to overcome the prior-established lesion-exacerbating effects of preventing astrocytic scar formation. Instead, injured axons are presented with neural and non-neural components of the spinal injury scar, including NG2+ OPCs, inflammatory cells and CSPGs, all of which are known blockers of regeneration141. Indeed, greater dieback of axons from the injury site was observed in this study108, in line with an advancing wall of inhibitory factors no longer contained in the fibrotic lesion core. Thus, rather than overturning an old dogma, this study used elegant genetic tools to demonstrate an important role for scar-forming astrocytes in tissue protection following traumatic spinal cord injury, supporting previous observations140 and confirming the early hypothesis postulated by Gopal D. Das: “If, by some means, glial scar formation could be completely eliminated, most of the atrophying axons still might not show regeneration, and the spinal cord would be continuously invaded by loose connective tissue and other foreign materials and organisms while undergoing a protracted degeneration”135.In addition, as discussed above, there is now increased appreciation of the multiple cell types, beyond astrocytes, which contribute to spinal injury scarring, together with extracellular and non-neural components. This renders the term glial scar an insufficient descriptor. The multifaceted nature of the scar should be considered when interpreting experimental approaches which prevent scar formation. For example, a number of transgenic loss of function experiments have been performed to specifically investigate the role of astrocytes following spinal cord injury, including formation of the glia limitans and continued presence in the chronic scar. Early evidence suggested that double knockout mice for the intermediate filament proteins GFAP and vimentin  develop a less dense glial scar, with greater haemorrhaging, fibrosis and presence of debris following lesion to the dorsal funiculus143. However, astrocyte ablation at chronic timepoints indicates that astrocytes themselves are necessary for maintaining tissue integrity in chronic injuries, as functional outcome is negatively affected by their removal 5 weeks post injury108. Though their contribution to extracellular signalling may have both beneficial and detrimental elements.Thus, it will be important to further characterise the molecular profile of the spinal injury scar at precise time points after injury, both for the cell types involved in scarring (e.g. Box 1) as well as the extracellular components. For example, recent proteomics studies of subacute (1-2 week) and chronic (8 week) spinal injury tissues have revealed differential expression of multiple growth factors and inhibitory and permissive ECM molecules at different post-injury stages50, requiring further study as to their specific roles in shaping the response to injury.Although astrocyte components of the spinal injury scar are well evidenced to be beneficial initially, they (and other scar elements) have been suggested to be detrimental at chronic post-injury stages145. Rather than focus on good versus bad, perhaps efforts would be best directed at understanding and targeting specific aspects of the scar to aid recovery. For example, it may be beneficial to target components of the scar which are non-permissive to regeneration or plasticity rather than removal of the astrocytes themselves, even at chronic time-points. Targeting diverse cell types and phenotypes, as well as extracellular and non-neural components should also be considered, as well as the timings of such interventions. These approaches will be discussed in the following section.Thus, the dual nature of the spinal injury scar has long been known, yet continues to be reviewed, revisited and reinterpretedCurrent experimental approaches for targeting the spinal injury scar attempt to either reduce scar formation, or to block inhibitory molecules associated with the scar, using a variety of surgical, pharmacological and genetic approaches. Some of these show promise for application in the clinic.146 and indeed expansion duroplasty is performed alongside decompressive durototomy in clinical evaulations147.Although preventing formation of the astrocytic component of the spinal injury scar impacts negatively on wound-healing (discussed above), as the mechanistic understanding of spinal injury scar pathology increases, a number of studies in preclinical rodent models have targeted mesenchymal or fibrotic-derived components in a bid to limit amplification of tissue damage. On a gross tissue scale, if the dura is breached, dural apposition and/or patching with another soft tissue material (duraplasty) is suggested to limit fibrotic and connective tissue deposition from meningeal-derived fibroblasts. Decompressive durotomy followed by dural allograft has been shown in rodent models to reduce scar formation and lesion volume, but if the dura is not replaced, lesion volume increases dramatically148. This highlights the potential for repurposing of epothilones and taxanes that are already used in cancer treatment149. Inhibiting collagen synthesis using the iron chelators BPY-DCA  and cyclic adenosine monophosphate 151 reduces fibrotic scarring and promotes neuroprotection and long-distance axon regeneration150. Treatment with clinically approved ion chelator deferoxamine and inhibition of lysl oxidase, another key collagen biosynthetic enzyme, also improves outcome after partial spinal transection injuries in rodents153.The fibrotic components of the scar can also be targeted pharmacologically. Systemic administration of the microtubule stabilizing antimitotic agents taxol or Epothilone B leads to reduced migration of scar-forming fibroblasts and suppression of extensive scar formation, enabling axon regeneration and functional recovery93. However, this is difficult to target therapeutically. If current studies of astrocyte phenotype in the mouse brain . Some of these are thought to augment pathology and extent of spinal injury scarring, while some directly inhibit the ability of neurons to overcome a scar environment and generate novel connectivity. Both are potential therapeutic targets.70. Daily intraperitoneal injections with a mouse monoclonal antibody against periostin from 4 days to 2 weeks after injury was shown to reduce the extent of tissue pathology and scarring, which led to functional improvements in sensorimotor tasks following contusion injury in mice70. Generation of recombinant anti-periostin will allow this promising strategy to be further tested. Similarly, a recent study found that pathology could be attenuated within the first 2 weeks following spinal cord injury in mice treated with an N-cadherin neutralizing antibody, which blocked an integrin and N-cadherin dependent interaction between extracellular type-1 collagen and astrocytes and significantly attenuated astrocytic scar formation54. In this study the rapid behavioural recovery observed supports a neuroprotective role for neutralising n-cadherin, though this was not assessed directly. As above, these studies suggest that early fibrosis is an important therapeutic target for improving outcome after spinal cord injury. Furthermore, if A1/A2 polarisation factors94 are conserved following spinal cord injury, a known inhibitor of C1q is chondroitin sulfate A156, the mono CS-4 sulfated GAG, which raises interesting questions regarding additional roles of ECM proteoglycans and their sulfation epitopes following injury. Thus, matrix properties are a valuable means to tap into the plasticity of cell responses.Periostin is a secreted ECM protein which has recently been implicated in contributing to scar formation, via propagating fibrosis and inflammatory signalling157, conditional sox9 ablation158 and knockout of N-acetylegalactosaminyltransferase-1, the enzyme which catalyses the addition of the first GalNAc residue onto the tetrasaccharide link between the core PG and GAG159. Reports of therapeutically-applicable pharmacological approaches which recapitulate these effects are currently lacking.CSPGs see Box  are know163. This effect has been replicated across multiple laboratories and in different species164, including mouse, rat, cat, and recently in primates165 and in a canine clinical model166. Furthermore, its use as an adjunct therapy can augment the benefits of other experimental therapeutics169. A gene therapy method of enzyme delivery, where host cells are themselves transduced to express the ChABC gene leads to extensive CS-GAG digestion, which results in reduced pathology and improved functional recovery following contusion injury to the thoracic170 and cervical172 spinal cord. Furthermore, widespread CSPG modulation achieved by viral delivery of ChABC promotes conversion of macrophages towards a pro-resolving M2 polarization state170 and drives an anti-inflammatory IL-10-mediated response173, which is likely to underlie reduced pathology. Thus, ChABC is a promising means to promote resolution of pathology as well as overcoming the inhibitory environment of the spinal injury scar. A recent study utilised a novel gene switch to enable controlled delivery of the ChABC gene and revealed that long term ChABC gene expression was required to elicit recovery of skilled reach and grasp functions, with recovery attributed to plasticity of descending systems172. Whether this viral ChABC approach will also have benefit when applied chronically is not yet established. However, recent work has demonstrated that a single injection of ChABC enzyme in the phrenic motor pool 1.5 years after unilateral cervical spinal cord injury was able to elicit rapid and robust recovery of respiratory function, restoring the ventilatory response to the paralysed hemidiaphragm174. Furthermore, chronic application of ChABC prior to transplantation of induced pluripotent stem cell-derived neural stem cells 7 weeks after a spinal compression injury led to reduced chronic-injury scarring, increased graft survival and improved limb function175. These studies highlight the potential for ChABC to unmask latent neuroplasticity and produce a microenvironment conducive to repair even within the chronic spinal injury scar.Enzymatic strategies targeting CSPGs are a promising approach for spinal cord repair, due to their ability to render the ECM more permissive to neuronal plasticity and connectivity. Removal of CS-GAGs by the chondroitinase ABC (ChABC) enzyme has been widely demonstrated to have beneficial effects in enhancing axonal regeneration and neuroplasticity and promoting functional recovery following experimental spinal cord injury176. ARSB perhaps represents a more attractive, and more readily translatable, therapeutic prospect than a bacterial enzyme such as ChABC and certainly warrants further investigation, particularly given recent findings of eliciting enhanced axon growth in the injured optic nerve177. However, whether ARSB could elicit robust modulatory effects within the spinal injury scar microenvironment, comparable to ChABC, remains to be determined. Given the specificity of ARSB for 4S motifs, it may not be capable of the multi-modulatory effects that have been demonstrated for ChABC which include immune modulation, neuroprotection and neuroplasticity179.Another enzymatic strategy that has recently been exploited for reducing CSPG inhibition is the mammalian enzyme Arylsulfatase B , which removes C4S moieties specifically from CS-GAGs. In addition to being utilized in enzyme-replacement therapy for human mucopolysaccharidosis VI, ARSB administration has now been shown in one study to promote increased axonal sprouting and functional locomotor recovery following compression spinal cord injury in the mouse180 and the non-invasive nature of this approach means it is a potential candidate for rapid translation. Thus, approaches to overcome the inhibitory actions of CSPGs show collective promise in enabling beneficial alterations to the ECM associated with the spinal injury scar with positive effects in eliciting some functional repair.Targeted modulation of CSPG receptor signalling via manipulation of the receptor PTPσ, has also proved to be a promising therapeutic prospect. The activity of the intracellular phosphatase domains of PTPσ are regulated via a conserved “wedge” structure which can occlude the catalytic domain, thus reducing phosphorylation activity and ability to signal downstream. Use of a membrane-permeable peptide mimetic of this wedge reduces PTPσ signalling following activation by ligands such as CSPGs. Systemic delivery of this peptide has been shown to enable recovery of locomotor and bladder function in rats following spinal contusion injury94, there may be further opportunity to modulate astrocyte function or phenotype following spinal cord injury. These approaches will likely evolve as new markers are identified for delineating reactive astrocytes and microglia in different phenotypic states181, with increasing availability of astrocyte and microglia cell-specific sequencing data108 and with powerful emerging tissue sequencing technology182. Alternative approaches which may indirectly modulate astrocyte phenotype are also emerging, such as grafting specific stem cell populations which can influence host tissue cellular responses and drive astrocyte transformation to a permissive phenotype132. Additionally, with increased appreciation for the role of ECM molecules in affecting pathology and plasticity of cellular responses, novel targets may be identified with new matrix biology technology183. Conversely, the study of scarring mechanisms and ECM components in organisms that are capable of CNS regeneration may lead to the identification of pro-repair targets. For example, differential regulation of collagen XII within the scar matrix is one factor contributing to the pro-regenerative phenotype in zebrafish, controlled by Wnt/B catenin signalling184. Whether Wnt/B-catenin-mediated collagen XII production can be harnessed to render the mammalian spinal injury scar more permissive is not yet known. Finally, consideration should be given to scar biomechanics125 when designing therapies. It may be important to understand how pharmacological manipulations affect mechanobiology and further provide appropriate mechanical signals to optimize repair.The majority of approaches aimed at manipulating the spinal injury scar for therapeutic benefit have focused on modifying scar-associated ECM and targeting the synthesis, production and signalling of CSPGs. With the identification of cell subtypes that have opposing actions on tissue pathology, such as A1 neurotoxic vs A2 reparatory astrocytes173. Conversely, altering intracellular mechanisms can alter the inhibitory environment (for example microtubule stabilization with taxol or epothilone B leads to reduced fibrotic scarring148). Similarly, astrocyte-immune cell interactions are bidirectional, where an increasingly proinflammatory environment induces extensive astrogliosis53 and in turn, activated astrocytes release pro-inflammatory cytokines, chemokines and CSPGs, which can influence the magnitude of the inflammatory response48. The spinal injury scar has both beneficial properties  and detrimental properties (limiting new growth and tissue repair). This may be attributable to opposing phenotypes of reactive glial cells that form the scar border, given recent evidence in other CNS pathologies94. However, also important to note are the opposing roles of the scar matrix which contains beneficial molecules, required for formation of the glia limitans  as well as molecules that are potent inhibitors of growth and neuroplasticity, such as CSPGs. Therapeutic strategies need to target detrimental aspects while preserving the beneficial properties of the spinal injury scar. Increased mechanistic understanding of the biological processes that propagate the non-resolving scar pathology is providing new therapeutic targets which may bring us closer to improving functional outcome following traumatic spinal cord injury.The spinal injury scar is multifaceted. It contains more than just a reactive glial component and should be considered as a whole, since there is a complex interplay between multiple different cell types , their intracellular and signalling changes, and the extracellular environment. These processes modulate and feedback on each other. Altering the environment, for example using CSPG modulation with chondroitinase, can increase neuronal regeneration-associated gene expression and transcriptional changes