Scarring In Mice Research Articles

The kinase inhibitory region of SOCS3 attenuates reactive astrogliosis and astroglial scar in mice after traumatic brain injury

Traumatic brain injury (TBI) leads to reactive astrogliosis that impedes neural repair/regeneration. It has been proven that SOCS3 attenuates astrocyte activation by inhibiting the JAK2-STAT3 pathway. However, whether the kinase inhibitory region (KIR) of SOCS3 can be directly applied to mediate astrocyte activation after TBI is not clear. The present study aimed at investigating the inhibitory effect of KIR on reactive astrogliosis and its potential neuroprotection after TBI insult. For this purpose, A TBI model was developed by the free impact of heavy objects in adult mice. KIR was linked to the TAT peptide (TAT-KIR) to facilitate cell membrane penetration and intracranially injected into the cerebral cortex adjacent to the TBI lesion. Then reactive astrogliosis, activity of JAK2-STAT3 pathway, neuron loss, and function deficit were observed. Our results showed a decrease in neuron loss and an improvement in neural function. Meanwhile, Intracranial injection of TAT-KIR in TBI mice demonstrated a reduction of GFAP-positive astrocytes as well as C3/GFAP double-labeled A1 reactive astrocytes. Western blot analysis illustrated that the activity of the JAK2-STAT3 pathway was significantly inhibited by TAT-KIR. We conclude that exogenous treatment TAT-KIR, through suppression of JAK2-STAT3 activity, inhibits TBI -induced reactive astrogliosis induced, thereby alleviating the loss of neurons and relieving the neural function deficit. This investigation suggests that TAT-KIR could be a potential therapeutic strategy for enhancing neural regeneration following

53. Wound Edge Inversion Delays Healing and Contributes to Dystrophic Scar Formation in a Mouse Model

Purpose: Management of dystrophic scars often requires surgical excision in the delayed periods as there are few reliable options to prophylax against the formation off dystrophic scars during physiologic or pathologic wound healing. This is in part because of limitations in the availability of genetically tractable small-animal models which can reliably generate the desired dystrophic tissue architecture. While using small animal models of wound healing is desired, the nature of murine dermal healing through contracture makes it poorly suited to the study of later wound sequalae such as dystrophic scars. We have previously demonstrated that tissue-edge inward folding mimicking the architecture of epibole mitigates contracture, restricts epithelialization, and prolongs wound opening. Here we demonstrate that murine epibolous wounds generate an environment of sustained inflammatory infiltration and proliferative granulation and ultimately generate consistent and reliable dystrophic scars in mice. Methods: Male C57BL/6J control (wt/wt) and diabetic (db/db) mice received bilateral 6mm excisional wounds. Each wound was stratified as either untreated control or chronic model. Chronic wounds were generated by incising skin flaps, which were sutured to the dermis side to create a folded skin edge. A first (acute) cohort was s were maintained for 14-days until all control wounds had closed and/or epithelialized and were harvested for flow cytometry for neutrophils and/or macrophages and/or histology for pan-keratin (epithelium) and alpha-smooth-muscle actin (myofibroblasts). A second (delayed) cohort was maintained until closure up to 63 days. Photographs were taken at time of sacrifice and samples were collected for H&E and Masson’s Trichrome Stain. Results: The epibolous wound environment maintained prolonged neutrophil-dominant inflammatory infiltrate up to 2-weeks with simultaneous enrichment of macrophage/monocyte populations (p<0.05). Epibolous wounds demonstrated reduced frequency of epithelialization as defined by pan-keratin signaling. In epibolous wounds without epithelialization, strong alpha-smooth-muscle actin signal was noted. When allowed to proceed to maturation, scars from epibole injuries remained significantly larger (p<0.05). This was true in both control and diabetic models. All wound-edge inversion injuries demonstrated gross and histologic evidence of dystrophic healing and collagen deposition compared to controls. Conclusion: Previously we demonstrated that wound-edge inversion to mimic epibole prolongs wound opening. Here we demonstrate that this translates to prolongs inflammation and a transition of murine healing to a more human-relevant granulation pattern. Furthermore, the delay of closure results in a consistent dystrophic scar without need for thermal, radiological, chemical or biological interference. Consequently, this simple surgical maneuver enhances our ability to study and intervene in the process of acute and chronic wound development and dystrophic scar formation in a histologically humanized model while retaining access to the genetic tractability of murine stock.

Neutrophil extracellular traps contribute to myofibroblast differentiation and scar hyperplasia through the Toll-like receptor 9/nuclear factor Kappa-B/interleukin-6 pathway.

Inflammation is an important factor in pathological scarring. The role of neutrophils, one of the most important inflammatory cells, in scar hyperplasia remains unclear. The purpose of this article is to study the correlation between neutrophil extracellular traps (NETs) and scar hyperplasia and identify a new target for inhibiting scar hyperplasia. Neutrophils were isolated from human peripheral blood by magnetic-bead sorting. NETs in plasma and scars were detected by enzyme-linked immunosorbent assays (ELISAs), immunofluorescence and flow cytometry. Immunohistochemistry was used to assess neutrophil (CD66B) infiltration in hypertrophic scars. To observe the entry of NETs into fibroblasts we used immunofluorescence and flow cytometry. We found that peripheral blood neutrophils in patients with hypertrophic scars were more likely to form NETs (p < 0.05). Hypertrophic scars showed greater infiltration with neutrophils and NETs (p < 0.05). NETs activate fibroblasts in vitro to promote their differentiation and migration. Inhibition of NETs with cytochalasin in wounds reduced the hyperplasia of scars in mice. We induced neutrophils to generate NETs with different stimuli in vitro and detected the proteins carried by NETs. We did not find an increase in the expression of common scarring factors [interleukin (IL)-17 and transforming growth factor-β (TGF-β), p > 0.05]. However, inhibiting the production of NETs or degrading DNA reduced the differentiation of fibroblasts into myofibroblasts. In vitro, NETs were found to be mediated by Toll-like receptor 9 (TLR-9) in fibroblasts and further phosphorylated nuclear factor Kappa-B (NF-κB). We found that IL-6, which is downstream of NF-κB, was increased in fibroblasts. Additionally, IL-6 uses autocrine and paracrine signaling to promote differentiation and secretion. Our experiments found that NETs activate fibroblasts through the TLR-9/NF-κB/IL-6 pathway, thereby providing a new target for regulating hypertrophic scars.

The serine proteases dipeptidyl-peptidase 4 and urokinase are key molecules in human and mouse scar formation

Despite recent advances in understanding skin scarring, mechanisms triggering hypertrophic scar formation are still poorly understood. In the present study, we investigate mature human hypertrophic scars and developing scars in mice at single cell resolution. Compared to normal skin, we find significant differences in gene expression in most cell types present in scar tissue. Fibroblasts show the most prominent alterations in gene expression, displaying a distinct fibrotic signature. By comparing genes upregulated in murine fibroblasts during scar development with genes highly expressed in mature human hypertrophic scars, we identify a group of serine proteases, tentatively involved in scar formation. Two of them, dipeptidyl-peptidase 4 (DPP4) and urokinase (PLAU), are further analyzed in functional assays, revealing a role in TGFβ1-mediated myofibroblast differentiation and over-production of components of the extracellular matrix in vitro. Topical treatment with inhibitors of DPP4 and PLAU during scar formation in vivo shows anti-fibrotic activity and improvement of scar quality, most prominently after application of the PLAU inhibitor BC-11. In this study, we delineate the genetic landscape of hypertrophic scars and present insights into mechanisms involved in hypertrophic scar formation. Our data suggest the use of serine protease inhibitors for the treatment of skin fibrosis.

A mouse model of placenta accreta spectrum

IntroductionPlacenta Accreta Spectrum (PAS) disorder is one of the leading causes of maternal morbidity and mortality due to uncontrollable hemorrhage. The greatest risk factor for development of PAS is prior uterine surgery, frequently cesarean delivery. Despite considerable clinical knowledge, animal models of PAS are lacking. To address this, we used two surgical approaches to create uterine scarring in peripartum and non-pregnant CD-1 mice. Il10−/− mice, with a pro-inflammatory phenotype were also studied. MethodsIn peripartum mice, a hysterotomy was performed to simulate a cesarean section. The second approach utilized endometrial curettage in non-pregnant mice. Sham-operated mice served as control. Following recovery, females were mated. On gestation day 16, pregnant females were euthanized, and the uterus was excised. Tissue was fixed, sectioned, and stained with H&E or cytokeratin immunohistochemistry. The cytokeratin-positive area extending beyond the trophoblast giant cells was measured by quantitative image analysis. Disruption of the circular (inner myometrium) smooth muscle was scored semi-quantitatively. ResultsIn surgically scarred mice, trophoblast invasion was deeper relative to control mice, regardless of surgical method. The myometrium in experimental mice showed significant disruption compared to sham controls. Results from CD-1 and Il10−/− mice were similar, with the latter showing more severe pathology. DiscussionWhile further refinement of surgical method is required, our data indicate that surgical uterine scarring in mice represents a promising model of PAS.

Cardiomyocytes from CCND2-overexpressing human induced-pluripotent stem cells repopulate the myocardial scar in mice: A 6-month study

BackgroundCardiomyocytes that have been differentiated from CCND2-overexpressing human induced-pluripotent stem cells (hiPSC-CCND2OE CMs) can proliferate when transplanted into mouse hearts after myocardial infarction (MI). However, it is unknown whether remuscularization can replace the thin LV scar and if the large muscle graft can electrophysiologically synchronize to the recipient myocardium. Our objectives are to evaluate the structural and functional potential of hiPSC-CCND2OE CMs in replacing the LV thin scar. MethodsNOD/SCID mice were treated with hiPSC-CCND2OE CMs (i.e., the CCND2OE group), hiPSC-CCND2WT CMs (the CCND2WT group), or an equal volume of PBS immediately after experimentally-induced myocardial infarction. The treatments were administered to one site in the infarcted zone (IZ), two sites in the border zone (BZ), and a fourth group of animals underwent Sham surgery. ResultsSix months later, engrafted cells occupied >50% of the scarred region in CCND2OE animals, and exceeded the number of engrafted cells in CCND2WT animals by ~8-fold. Engrafted cells were also more common in the IZ than in the BZ for both cell-treatment groups. Measurements of cardiac function, infarct size, wall thickness, and cardiomyocyte hypertrophy were significantly improved in CCND2OE animals compared to animals from the CCND2WT or PBS-treatment groups. Measurements in the CCND2WT and PBS groups were similar, and markers for cell cycle activation and proliferation were significantly higher in hiPSC-CCND2OE CMs than in hiPSC-CCND2WT CMs. Optical mapping of action potential propagation indicated that the engrafted hiPSC-CCND2OE CMs were electrically coupled to each other and to the cells of the native myocardium. No evidence of tumor formation was observed in any animals. ConclusionsSix months after the transplantation, CCND2-overexpressing hiPSC-CMs proliferated and replaced >50% of the myocardial scar tissue. The large graft hiPSC-CCND2OE CMs also electrically integrated with the host myocardium, which was accompanied by a significant improvement in LV function.

A novel model of humanised keloid scarring in mice

Treatments for keloid scarring are a major challenge to scientists and physicians for their unknown aetiology. Although several models, including monolayer cell culture to tissue-engineered models, were developed, further research on keloid has more or less been hindered by the lack of appropriate animal models. Because these aberrant scars are specific to humans, we obtained human normal and keloid skin tissues and isolated dermal fibroblasts from them. Cell morphology, growth and immunohistochemical staining of myofibroblastmarker α-SMA were examined, and the cell medium of 2-hour culture and 24-hour culture was implanted on the back of nude mice. The cell medium of 2-hour culture and 24-hour culture was also analysed by a protein array for the detection of distinction in inflammatory factors. We showed that keloid fibroblasts had similar morphology and growth compared to normal skin fibroblasts, but the α-SMA expression was obviously up-regulated. After 6 weeks, mice of the 2-hour keloid-derived culture medium group exhibited keloid-like hypertrophic nodules macroscopically, while mice of 24-hour keloid-derived culture medium group were similar to normal skin. Histological findings confirmed that the reconstituted skin tissues had the typical features of human keloids. The protein array data revealed that RANTES were involved in humanised fibrotic occurrence in mice, also suggesting they were important modulators of this inflammatory event. This novel model might help to understand the key events that result in the formation of these abnormal scars and provide new therapeutic options.

Baicalein attenuates hypertrophic scar formation via inhibition of the transforming growth factor-β/Smad2/3 signalling pathway.

Hypertrophic scars (HPSs) are characterized by excessive fibrosis associated with aberrant function of fibroblasts. Currently no satisfactory treatment has been developed. To investigate the effect of baicalein on HPSs and its underlying mechanisms. Baicalein was administered intradermally (10 μmol L(-1) in 100 μL sterile saline plus 10% dimethylsulfoxide) to mechanical-load-induced scars in mice once a day for 14 or 28 days. Histological and immunohistochemical studies were performed to evaluate scar hypertrophy and the function of fibroblasts. Human HPS-derived fibroblasts (HSFs) were determined by immunofluorescence study, collagen gel contraction assay and wound-healing assay. Also, protein expression of the transforming growth factor (TGF)-β signalling pathway was detected using Western blotting. Lastly, a molecular docking study and kinase binding assay were conducted in search of the potential interaction between baicalein and activin receptor-like kinase (ALK)5. Baicalein treatment significantly attenuated HPS formation and collagen deposition in a mechanical-load-induced mouse model. Baicalein also inhibited the proliferation and activation of fibroblasts in vitro and in vivo. Furthermore, baicalein impaired the contractile and migration ability of HSFs. Protein expression investigation revealed that baicalein had an inhibitory effect on TGF-β/Smad2/3 signalling. Such bioactivity of baicalein may result from its selective binding to the ATP-binding pocket of ALK5, as suggested by the molecular docking study and kinase binding assay. Baicalein could serve as a promising agent for treatment of HPSs and a selective ALK5 inhibitor.

Steroids Alone or as Adjunctive Therapy with Doxycycline Fail To Improve Oviduct Damage in Mice Infected with Chlamydia muridarum

In women, Chlamydia trachomatis can ascend from the cervix to the fallopian tubes, where an overly aggressive host inflammatory response can cause scarring that leads to chronic pelvic pain, infertility, or ectopic pregnancy. Although screening and treatment programs for women have resulted in decreased rates of sequelae, morbidities associated with oviduct scarring continue to occur. Since corticosteroids have anti-inflammatory and antifibrotic effects, we tested the ability of dexamethasone to inhibit inflammation and prevent oviduct scarring in mice genitally infected with Chlamydia muridarum. The administration of 1 or 2.5 mg/kg of body weight of dexamethasone on days 7 to 21 of infection resulted in reduced accumulation of inflammatory cells in the oviducts compared to that in controls. However, a concomitant increase in bacterial burden was observed, and chronic oviduct disease was not reduced. Adjunctive administration of a prolonged (21-day) or short (3-day) course of dexamethasone in combination with the antibiotic doxycycline also failed to reduce chronic oviduct pathology compared to antibiotic treatment alone. Steroids administered alone or adjunctively with antibiotics failed to prevent oviduct damage in this murine model of C. trachomatis infection.

A Dual AP-1 and SMAD Decoy ODN Suppresses Tissue Fibrosis and Scarring in Mice

The transforming growth factor-β (TGF-β) signaling pathway promotes tissue fibrosis and scarring through SMAD (small mothers against decapentaplegic)-dependent and SMAD-independent mechanisms. However, inhibition of SMAD-mediated signal transduction alone induces an excessive inflammatory response that impairs the antifibrotic effects of TGF-β inhibitors. In this study, we designed and characterized a dual-functional transcription activator protein 1 (AP-1) and SMAD decoy oligodeoxynucleotide, antifibrosis oligodeoxynucleotide 4 (AFODN4) in vitro and in vivo. AFODN4 binds directly to recombinant AP-1 and SMAD with high affinity. AFODN4 significantly inhibited the DNA-binding and transcriptional activities of both AP-1 and SMAD, as well as the production of fibrotic mediators stimulated by TGF-β1 or TGF-β2 in L929 murine fibroblasts. Local administration of AFODN4 significantly inhibited fibrosis associated with acute dermal wounds in mice. Intriguingly, AFODN4 inhibited AP-1-mediated production of proinflammatory mediators, which can be caused by blockage of SMAD alone in vitro and in vivo. Collectively, these findings suggest that dual inhibition of SMAD and AP-1 signaling by AFODN4 is a useful strategy for the development of new antifibrotic agents.

Absence of the Adenosine A2A Receptor Attenuates Hypertrophic Scarring in Mice

Hypertrophic scar following cutaneous wounding is the result of the body overproducing collagen, and it has been shown that the activation of the adenosine A(2A) receptor promotes tissue repair, wound healing, and extracellular matrix production. In this study, we explored the role of adenosine receptor in hypertrophic scarring in adenosine A(2A) receptor knockout mice models. Mechanical stress was applied to a healing wound to produce hypertrophic scars in adenosine A(2A) receptor knockout mice and wild-type controls. Total scar areas were evaluated after routine hematoxylin and eosin and picrosirius red staining and hydroxyproline content measured colorimetrically. Expression of transforming growth factor-β in scar tissues was measured using the Western blotting method. Compared with the wild-type control group, adenosine A(2A) receptor knockout mice showed significantly less thickness, smaller cross-sectional area, and significantly lower hydroxyproline levels and transforming growth factor-β levels. These results demonstrate that adenosine A(2A) receptors play an active role in the pathogenesis of dermal fibrosis and suggest a novel therapeutic target in the prevention and treatment of hypertrophic scarring.

Antagonism of CCL5 chemokine receptors during regression of liver fibrosis is associated with an intrahepatic shift towards pro-inflammatory macrophages

Aims: Chemokines mediate the progression but also the regression of liver fibrosis. We could recently show that antagonism of the chemokine CCL5 with Met-CCL5 accelerates the regression of liver fibrosis (Berres et al. JCI 2010). Here we aimed to investigate the underlying mechanisms for the faster resolution of liver scarring in this model. Methods: C57BL/6 mice were treated with CCl4 for eight weeks to induce severe fibrosis. At peak of fibrosis (3 days after the last CCl4 injection), mice received either Met-CCL5 (10µg/day) or vehicle for the next 7 days. Mice were sacrificed 6 and 10 days after the last CCl4 injection and analyzed for their fibrosis and immune phenotype. Results: Both groups of animals (Met-CCL5 and vehicle treated) displayed a regression of liver fibrosis. However, the regression was accelerated in Met-CCL5 treated mice as evidenced by a lower Sirius red positive area and reduced hydroxyproline content at day 10 (both P < 0.05). This faster resolution of liver scarring was associated with a higher number of intrahepatic Gr1+ (Ly6C+) monocytes at all investigated time points (P < 0.05). Furthermore, the frequency of iNOS and TNF-alpha positive macrophages was increased in Met-CCL5 treated mice compared to vehicle treated mice. The shift to a more pro-inflammatory intrahepatic macrophage subset was confirmed by RT-PCRs of total liver tissue. Compared to vehicle treated mice, animals injected with Met-CCL5 displayed higher mRNA levels of iNOS (P < 0.05), but lower mRNA levels of IL–10 (P < 0.01) and arginase (P=0.2). Conclusions: The faster resolution of liver scarring in mice treated with an antagonist of CCL5 receptors is strongly associated with a shift of intrahepatic macrophage subsets to a pro-inflammatory phenotype. The results confirm earlier findings that monocyte/macrophage subsets differentially contribute to progression and resolution of liver fibrosis and that this balance can be successfully modulated by chemokine antagonists.

Suppression of Injury-Induced Conjunctiva Scarring by Peroxisome Proliferator-Activated Receptor γ Gene Transfer in Mice

To examine the effects of introduction of the adenoviral peroxisome proliferator-activated receptor (PPAR)-gamma gene on postinjury conjunctival scarring in mice. Its effects on fibrogenic reaction of cultured human subconjunctival fibroblasts (hSCFs) were also evaluated. The effects of PPARgamma gene introduction on expression of type I collagen, fibronectin, and connective tissue growth factor (CTGF) in hSCFs were examined. A circumferential incision was made in the equatorial conjunctiva of the right eye of generally anesthetized adult C57BL/6 mice (n = 72). PPARgamma cDNA-expressing adenoviral vector was topically applied; the control eye received nonfunctioning adenoviral vector. At 2, 5, 7, and 14 days (each, n = 18), the eyes were processed for histologic or immunohistochemical examination to evaluate tissue scarring. Expression of type I collagen and growth factors was evaluated by real-time reverse transcription-polymerase chain reaction in 32 eyes from control and treatment groups. PPARgamma overexpression suppressed type I collagen, fibronectin, and CTGF in cultured hSCFs at the mRNA or protein level. In vivo experiments showed that PPARgamma gene introduction suppressed monocyte/macrophage invasion, generation of myofibroblasts, and mRNA upregulation of cytokines/growth factors and collagen Ialpha2 chain (Col 1A2) in healing conjunctiva. PPARgamma gene transfer suppresses the fibrogenic reaction in hSCFs as well as the injury-induced scarring of conjunctival tissue in mice, suggesting the effectiveness of this strategy in preventing excess scarring after filtration surgery. The mechanism may include suppression of activation of fibroblasts and reduction of macrophage invasion.

Postinfarction Myocardial Scarring in Mice: Molecular MR Imaging with Use of a Collagen-targeting Contrast Agent

To prospectively evaluate a gadolinium-based collagen-targeting contrast agent, EP-3533, for in vivo magnetic resonance (MR) imaging of myocardial fibrosis in a mouse model of healed myocardial infarction (MI). All procedures were performed in accordance with protocols approved by the animal care and use committee. MI was induced in eight mice by means of occlusion of the left anterior descending coronary artery followed by reperfusion. Four MR examinations were performed in each animal: one examination before, one examination 1 day after, and two examinations 6 weeks after the MI. For the latter two examinations, electrocardiographically gated inversion-recovery gradient-echo MR images were acquired before and serially (every 5 minutes) after the intravenous injection of either gadopentetate dimeglumine or EP-3533. The image enhancement kinetic properties of the postinfarction scar, normal myocardium, and blood were compared. Dynamic T1-weighted MR imaging revealed the washout time constants for EP-3533 to be significantly longer than those for gadopentetate dimeglumine in regions of postinfarction scarring (mean, 194.8 minutes +/-116.8 [standard deviation] vs 25.5 minutes +/- 4.2; P < .05) and in normal myocardium (mean, 45.4 minutes +/- 16.7 vs 25.1 minutes +/- 9.7; P < .05). Findings on postmortem histologic sections stained for collagen correlated well with EP-3533-enhanced areas seen on inversion-recovery MR images. Fifty minutes after EP-3533 injection, the postinfarction scar tissue samples, as compared with the normal myocardium, had a twofold higher concentration of gadolinium. Use of the gadolinium-based collagen-targeting contrast agent, EP-3533, enabled in vivo molecular MR imaging of fibrosis in a mouse model of healed postinfarction myocardial scarring.

Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis

Hypertrophic scars occur following cutaneous wounding and result in severe functional and esthetic defects. The pathophysiology of this process remains unknown. Here, we demonstrate for the first time that mechanical stress applied to a healing wound is sufficient to produce hypertrophic scars in mice. The resulting scars are histopathologically identical to human hypertrophic scars and persist for more than six months following a brief (one-week) period of augmented mechanical stress during the proliferative phase of wound healing. Resulting scars are structurally identical to human hypertrophic scars and showed dramatic increases in volume (20-fold) and cellular density (20-fold). The increased cellularity is accompanied by a four-fold decrease in cellular apoptosis and increased activation of the prosurvival marker Akt. To clarify the importance of apoptosis in hypertrophic scar formation, we examine the effects of mechanical loading on cutaneous wounds of animals with altered pathways of cellular apoptosis. In p53-null mice, with down-regulated cellular apoptosis, we observe significantly greater scar hypertrophy and cellular density. Conversely, scar hypertrophy and cellular density are significantly reduced in proapoptotic BclII-null mice. We conclude that mechanical loading early in the proliferative phase of wound healing produces hypertrophic scars by inhibiting cellular apoptosis through an Akt-dependent mechanism.

078 Mechanical Strain Induces Hypertrophic Scars in Mice by Reducing Stromal Cell Apoptosis

Intro: Pathophysiology of hypertrophic scar formation remains unclear, potentially involving mechanical strain, burns, or infection. We sought to specifically examine the role of mechanical strain in hypertrophic scars. To do so, we developed and characterized a novel murine model which uses mechanical strain to produce hypertrophic scars in mice. Methods: Paired incisions were created on C57BL6 mice (n = 20). After closure, mechanical strain was applied across one wound using a novel device; the other wound was a control. Starting at day 3, mechanical strain was increased every other day over 4 wks. Wounds were harvested each week, and examined histologically using Sirius red, DAPI, BrdU for proliferation, and caspase 3 for apoptosis. Results: Strained wounds showed features of hypertrophic scars: raised borders, loss of rete pegs and adnexal structures in the epidermis overlying scars, blood vessels that were perpendicular and fibrillary collagen that was parallel to skin surface. Features persisted beyond 6 months. Wound collagen deposition in strained wounds increased 6‐fold at 1 wk and over 12‐fold at 2–4 wks, in parallel to an increase in number of stromal cells within scar (28‐fold). This resulted in an increase in number of cells per unit area of collagen (over 42%). In all wounds, there was no significant difference in proliferation. There was a significant reduction in blood vessel (4‐fold) and fibroblast (3‐fold) apoptosis at 1–4 wks in strained wounds. Conc: Mechanical strain alone is sufficient to produce hypertrophic scars in this model that are indistinguishable from human scars. Notably, hypertrophic scars appear to result from a marked reduction in apoptosis, rather than from a significant proliferation and upregulation of cellular collagen deposition, validating previous in vitro studies.

Involvement of CD8+ T-cells in exacerbation of corneal scarring in mice

Purpose. To determine the specific immune responses involved in the exacerbation of corneal scarring induced by HSV-1 in gK vaccinated mice.Materials and methods. BALB/c mice were vaccinated with HSV-1 glycoprotein K (gK) and ocularly challenged with HSV-1. Infiltration into the cornea of T cells and macrophages was monitored by immunocytochemistry, and the effect of depletion of CD4+ T-cells, CD8+ T-cells, or macrophages on corneal scarring was determined.Results. Following ocular challenge, CD4+ and CD8+ T-cells and macrophages were more abundant in the corneas of gK-vaccinated mice than in the corneas of mock vaccinated mice. Depletion of CD8+ T-cells, but not of CD4+ T-cells or macrophages, reduced the severity of corneal scarring in gK-vaccinated mice.Conclusions. We have shown that gK vaccination causes an overall increase in T cells and macrophages in the cornea after ocular HSV-1 challenge. The immunopathology induced by gK vaccination appears to be related to CD8+ T-cell activity, as depletion of these cells, but not other immune cells, reduced corneal scarring.