Macrophages are critical mediators of injury-associated corneal hemangiogenesis (HA) and lymphangiogenesis (LA). Yet, molecular regulators of the hem- and lymphangiogenic potential of corneal wound macrophages are poorly understood. Using two different mouse models of acute  and chronic  corneal injury, here we identified distinct functions of early- versus late-phase corneal wound macrophages in corneal HA and LA. Whereas early-phase wound macrophages are essential for initiation and progression of injury-mediated corneal HA and LA, late-phase wound macrophages control maintenance of established corneal lymphatic vessels, but not blood vessels. Furthermore, our findings reveal that the hem- and lymphangiogenic potential of corneal wound macrophages is controlled by the type of the corneal damage. Whereas perforating corneal incision injury induced primarily wound macrophages with lymphangiogenic potential, corneal suture placement provoked wound macrophages with both hem- and lymphangiogenic potential. Our findings highlight a previously unrecognized injury-context dependent role of early- versus late-phase corneal wound macrophages with potential clinical impact on therapy development for sight-threatening corneal neovascular diseases. Corneal wound healing usually proceeds without physiological angiogenesis. However, in case of severe corneal injury the corneal “(lymph)angiogenic privilege” is overpowered by a massive upregulation of pro-angiogenic stimuli leading to an ingrowth of blood vessels (BV) and lymphatic vessels (LV) from the limbal arcade towards the corneal center . Although these neovessels serve to supply cells of the immune system, growth factors and cytokines and in turn mediate their clearance to support corneal wound healing5, corneal neovascularization is mostly considered undesirable as ingrowth of BV can interfere with corneal transparency, and lead to lipid deposition and hemorrhage through immature capillaries3. LV are clinically invisible and therefore do not noticeably impair corneal transparency but are considered the main risk factor for corneal transplant rejection7. Corneal LV have additionally been linked to development of ocular surface diseases like dry eye disease and ocular allergy9. However, we have recently also demonstrated beneficial functions of corneal LV: comparable to LV in cutaneous11 or intestinal inflammation12, corneal LV may support the resolution of persistent inflammation and additionally may be involved in the regulation of corneal edema13. Therefore, dissecting the cellular and molecular mechanisms that orchestrate the hem- and lymphangiogenic balance in the injured and regenerating cornea is crucial for the development of efficient therapeutic approaches for the treatment and prevention of corneal neovascular diseases, but also to promote corneal repair responses.The cornea as the front part and major refractive element of the eye, is physiologically avascular and alymphatic. This corneal avascularity is essential for transparency and is actively maintained by several antiangiogenic mechanisms16. It is established that macrophages are able to secrete vascular endothelial growth factor (VEGF)-A, VEGF-C, and VEGF-D that promote vascular endothelial proliferation17. Our group and others have previously demonstrated that depletion of macrophages decreases angiogenesis in experimental corneal neovascularization14 and leads to impaired corneal wound healing in epithelial debridement and corneal transplantation models19.Substantial evidence indicates that macrophages are essential mediators of corneal hemangiogenesis (HA) and lymphangiogenesis (LA) after injury22. Similar dynamics could be involved in corneal angiogenesis23. However, it is currently unclear whether macrophages exert different hem-/lymphangiogenic potency during subsequent stages in different settings of corneal damage and how macrophage-mediated angiogenesis supports the corneal repair response. In addition, it is unknown whether macrophages play a role in the maintenance of neovascular structures, which is of particular clinical interest in patients with corneal neovascularization usually presenting with already established corneal neovessels. Thus, in this study we aimed to analyze macrophage dynamics during the corneal inflammatory response after injury and to study the specific function of macrophages during corneal BV and LV initiation, progression, maintenance and regression using phase-restricted depletion of macrophages in subsequent phases after injury. For this purpose, we made use of two corneal injury models in mice: a perforating corneal incision model and the well-established corneal suture model. Both models differ in the kinetics, quality, and quantity of corneal neovascularization. In the incision model a central perforating linear incision disrupting all corneal layers is placed in the corneal center. We have previously shown that this type of injury leads to an isolated growth of LV, but not BV13. This lymphangiogenic response is resolved within 4 weeks following injury. Therefore, incision injury is considered as an acute corneal LA model13. By contrast, intrastromal suture placement leads to a prolonged chronic injury response. Here three figure-of-eight nylon sutures are placed intrastromally in the peripheral cornea equidistant from the limbus, which leads to epithelial and stromal injury at the site of suture placement24. Sutures are left in place for 14 days which leads to a strong inflammatory response as a result of foreign body reaction. Previous work has determined the time course of HA and LA in this model: BV and LV grow into the cornea simultaneously and peak on the day of suture removal. The subsequent vessel regression is faster for LV than for BV, as LV are completely regressed by 6 months, while BV can be detected up to 8 months after suture removal25.Moreover, our group has previously demonstrated that the angiogenic potential of macrophages changes during the progression of skin wound healing: we have shown that especially early stage macrophages recruited in the first hours and days after injury have non-redundant functions for the induction of vascular sprouts and the overall progression of proper skin wound healing, while late stage macrophages rather exert functions on collagen fibril crosslinking and extracellular matrix consolidation25, a characterization of the interrelationship between macrophage function and vascular growth at different time points after injury is lacking and was the aim of this study.Comparing these different injury models allowed to analyze the function of macrophages in HA/LA in acute versus chronic injury responses. Although both models (incision model and suture model) have previously been characterized in their vascular responses after injury13, whereas the well-established model of corneal suture placement, which is considered as a chronic injury model, results in parallel growth of corneal BV and LV25. In both models the inflammatory macrophage kinetics have not been characterized so far.Despite the common association of macrophages with corneal neovascularization, the time-specific function of macrophages in the process of inflammatory corneal HA/LA and wound healing is unknown. To dissect the role of macrophages in inflammatory corneal HA/LA, we made use of two different mouse models of corneal injury: the corneal incision model and corneal suture model. The incision model, which is considered as an acute injury model, leads to transient and isolated LA without concurrent HATo correlate LA and macrophage infiltration after incision injury, we compared the corneal area covered by LV and macrophages at different time points to uninjured controls Fig. . After i25. The amount of BV was still significantly elevated at day 56 after suture placement, which corresponds to six weeks after suture removal . By contrast, LV were mostly regressed at this time point  and were no longer significantly elevated compared to controls already from day 28 onwards . Similarly to the quickly developing and relatively long lasting vascular response, macrophage numbers increased quickly after suture placement and peaked on day 14 , when sutures were removed  Fig. . Intrastved Fig. . After sIn both corneal injury models angiogenesis and macrophage infiltration followed similar kinetics and correlated in time, indicating an interrelation between macrophages and vascular growth. We therefore made use of the different inflammatory and vascular kinetics in both models to analyze the proangiogenic properties of macrophages in steady state and after corneal injury.26. To analyze whether the steady state maintenance of the limbal blood and lymphatic vasculature in Balb/c mice, which are frequently used in corneal neovascularization models29, also depends on the presence of macrophages, we investigated the impact of macrophage depletion on the resting corneal vasculature in this mouse strain  Fig. , while dSimilarly to the incision model, depletion of early stage macrophages after corneal suture placement markedly reduced corneal LA  Fig. . MidstagIn intrastromal suture-placement both macrophages and neovessels persist long after the inflammatory stimulus is removed. In contrast to acute incision injury angiogenesis is not transient but persistent. To assess whether macrophages in this long-lasting inflammatory infiltrate are involved in maintenance of corneal vessels after removal of the initial stimulus, we performed macrophage depletion in late stages directly after suture removal starting from on day 14 Fig. , as wellIn contrast to corneal incision injury (which induces only LV but not BV growth) intrastromal suture placement leads to the simultaneous outgrowth of BV and LV. We therefore analyzed the contribution of macrophages to the initiation, progression, maintenance, and regression of blood vessels after this type of chronic injury. In this setting of corneal injury, ablation of early stage macrophages directly after suture placement strongly reduced the extent of inflammatory HA  Fig. , while mIn order to better characterize the inflammatory nature and involved macrophage phenotypes in the injury models used in this study, we performed real-time PCR analyses of injured corneas on day 7 after incision and suture injury Fig. . Expressin vitro , inflammatory macrophages appear to sustain lymphatic neovessel stability after chronic injury, but not after acute injury where LV begin to regress promptly with the progression of wound healing. Furthermore, our results demonstrate that the hemangiogenic potential of early stage corneal wound macrophages strongly depends on the type of injury.In this study we demonstrated that macrophage infiltration and macrophage-mediated corneal angiogenesis follow different dynamics during diverse settings of corneal injury. We identified distinct functions of early- versus late-phase wound macrophages after corneal injury and showed that the contribution of wound macrophages to the vascular repair response varies during healing progression. While early stage wound macrophages are essential for initiation and progression of both corneal HA and LA, late stage wound macrophages promote HA and LA to a far lesser extent but are involved in the maintenance of established corneal LV, but not BV after injury. These results unravel distinct and phase specific roles for wound macrophages in both vessel progression and maintenance in the injured cornea.13. By contrast, a chronic foreign body reaction during intrastromal suture placement may require the ingrowth of BV to ensure adequate influx of immune cells, growth factors, and nutrients. Comparing both injury models, it is likely that milder forms of injury only induce the outgrowth of LV to promote wound healing, while concurrent HA is required for proper healing of more severe injuries. Severe injuries might induce HA e.g. to accelerate the healing response or to prevent the transmission of infections to the inner eye, indicating that under these compromised conditions corneal transparency is immolated for the safety of the entire eye. In line with this, isolated LA is also observed in other corneal surface disease models like ocular allergy and dry eye9, whereas, to our knowledge, no corneal injury model displaying isolated HA exists.Both injury models analyzed in this study display different vascular responses: acute corneal incision injury leads to transient and isolated outgrowth of LV. By contrast, intrastromal suture placement stimulates both HA and LA, and both vessel types persist for a long time even when the inflammatory stimulus has already been removed. These observations demonstrate that the corneal hem- and lypmphangiogenic response varies depending on the type of injury. Although perforating incision injury results in a disruption of corneal integrity, the wound is quickly closed with epithelium, the anterior chamber is restored within 24 hours, and inflammation is quickly resolved. In this setting of corneal injury, only LV but not BV may be required for the progression of wound healing and the resolution of inflammation and edema32. Although also neutrophils34 and epithelial cells as well as keratocytes are considered major drivers of corneal angiogenesis due to their secretion of e.g. VEGF, PDGF, FGF and matrix metalloproteinases35, macrophages appear to have non-redundant pro-angiogenic functions in vessel progression, as our data demonstrate that early macrophage depletion almost completely abrogated BV and LV sprouting in our injury models.In previous studies macrophages were shown to express the hem- and lymphangiogenic growth factors VEGF-A, VEGF-C and VEGF-D after corneal injury13. These data indicate that macrophages are involved in the regulation of corneal volume and fluid homeostasis, arguably via the induction of LV. Indeed, other reports outside the eye have demonstrated similar functions for macrophages37. Late state depletion of macrophages when LV structures were already established did not influence LA after corneal incision. In this model LV usually quickly regress within 4 weeks after injury without further intervention, as soon as corneal edema is resolved13. As LA is transient in the incision injury model, the lymphangiogenic response after injury might not involve a process of vessel maturation. In late stage LV likely have already proceeded to regress and might not depend on macrophage-derived growth factors supporting their maintenance. Additionally, as resolution of edema and LA appear to correlate13, corneal edema rather than inflammation might be the driving force for LA in later stages in this injury model, in a process of flow-induced LA38. With proceeding resolution of edema, a decline in intravascular flow may therefore initiate LV regression independent of macrophages.Phase-specific macrophage depletion after acute incision injury demonstrated that corneal LA in the early phase after injury depends on the presence of prolymphangiogenic macrophages, as early macrophage depletion significantly reduced corneal LA. We have previously shown that corneal incision injury leads to profound corneal edema and that injury-associated corneal LA is involved in the regulation of corneal edemaOn the contrary, in suture placement a long-lasting inflammatory response appears to drive the angiogenic response and the maintenance of vascular structures. After suture placement, we observed a long-lasting macrophage infiltrate, which persisted even after the sutures were removed. However, from our experiments it is still unclear whether the initial inflammatory macrophage infiltrate is long-lived due to a lack of pro-apoptotic signals or if this observation is a result of ongoing recruitment, or local macrophage proliferation. This dense persisting macrophage infiltrate might account for the induction of HA in addition to LA in this model, assuming that the induction of HA requires higher levels of inflammation and macrophage-derived growth factors when compared to LA. However, the gene expression experiments performed in this study let us conclude that HA might also be mediated by a different subset of macrophages activated after suture placement.Our analyses revealed that in the suture model, type 1 and type 2 immunity is involved, whereas the predominant immune phenotype in the incision model seems to be type 2. Furthermore, we have shown that VEGF-A is predominantly induced in IFNγ/LPS-activated but not in IL-4/IL-13-activated macrophages, whereas VEGF-C and VEGF-D expression is induced in both macrophage subsets. Therefore, we conclude that incision injury is more associated with type 2 immunity and expression of VEGF-C and VEGF-D (but not VEGF-A), which selectively induces LA. By contrast, suture injury is associated with type 1 and type 2 immunity and expression of VEGF-A, VEGF-C and VEGF-D, resulting in HA as well as LA. Therefore, our findings indicate that the hem- and lymphangiogenic potential of corneal wound macrophages might be controlled by the type of the corneal damage. Certainly the discrimination between different macrophage subsets and activation states in acute versus chronic corneal injury requires further research.20. Also in the cornea, macrophages /macrophage derived factors may be particularly relevant in the sprouting phase of corneal HA, whereas BV maintenance and pruning may proceed independently of macrophages. It is well-established that vessel formation and maintenance are differently regulated41, which may account for the observed differences in the diverse phases after injury in our study. Otherwise, these results may be explained not by a change in macrophage proangiogenic potential but a decrease in growth factor sensitivity in endothelial cells. During vessel progression in early stage, endothelial cells may rely more on growth factors and may be more sensitive to growth factor deprivation than in later stages. Previous studies have shown that corneal neovessels are quickly covered by pericytes as they mature42, which abrogates VEGF responsiveness and promotes the transition into a mature and quiescent state45. In this regard, numerous studies e.g. in tumor angiogenesis models have shown that lack of pericyte coverage is accompanied by increased sensitivity to VEGF withdrawal47.In our experiments of phase-specific macrophage depletion during suture-induced corneal neovascularization, depletion had different outcomes regarding HA and LA. After macrophage depletion LV were considerably reduced at all time points analyzed, while the effect on BV decreased with time. Depletion in early stage following injury resulted in the strongest reduction of corneal HA, whereas depletion at later time points decreased HA to a far lesser extent. In advanced stage, 6 weeks after suture removal, HA was no longer affected by macrophage depletion. Thus, the effect of macrophage depletion in our experiments on HA decreased with time and proceeding vessel maturation. Early stage macrophages infiltrating the cornea directly after injury therefore appear to possess the highest hemangiogenic potential. In line with this, our previous work in skin wound healing demonstrated that early and midstage macrophages are essential for the induction of vascular sprouts and vessel stability, whereas late stage macrophages seem to be dispensable for HA48, macrophages have also been postulated to be directly involved in LV formation. Several groups have observed macrophages integrating into LV like structures and to de novo synthesize vessel like structures52, possibly by a process of transdifferentiation into lymphatic endothelial cells49. This process of direct integration of macrophages into LV might account for the stronger effect of macrophage depletion on LV than BV. However, the integration of macrophages into LV is still under discussion, as reports using lineage tracing have excluded macrophages as source of lymphatic endothelial progenitors both during embryogenesis and experimentally induced LA e.g. in skin53. Nevertheless, macrophages are key regulators of LA, although the precise mechanisms are not completely understood. Moreover, apart from their influence on LV sprouting after injury, macrophages in the cornea were previously reported to sustain the limbal lymphatic vasculature in the resting cornea, at least in C57Bl/6 mice26. In our current experiments we were not able to detect significant changes in the limbal blood and lymphatic vasculature following macrophage depletion in Balb/c mice, which may account for strain specific differences in corneal macrophages or LV55. Indeed, it has been shown that corneas of Balb/c mice contain fewer numbers of resident macrophages and possess discontinuous limbal lymphatic vessels54, which might be responsible for the observed differences in vessel maintenance under steady state conditions.In suture-induced neovascularization, corneal LV appeared to be more dependent on the presence of macrophages than BV. Early and midstage depletion strongly reduced LA, indicating that corneal macrophages are essential for LV progression arguably by secretion of lymphangiogenic factors. In late and advanced stage, macrophage depletion still affected LA and led to accelerated regression of LV. The fact that late stage LV are sensitive to macrophage depletion, indicates that LV permanently rely on macrophages/macrophage-derived growth factors, although the specific molecular factors leading to lymphatic endothelial stabilization have not been identified so far. Apart from the secretion of VEGF-C and -D that bind to VEGFR-3 on lymphatic endothelial cells, which promotes cell proliferation, migration and survival9. Additionally, these strategies could be implemented to reduce graft rejection after corneal transplantation, as we and others have previously shown that especially LV mediate immune rejection episodes after corneal grafting7.In summary, our results imply that macrophage-targeted treatment strategies may only be sufficient to attenuate BV progression in the cornea but may not provide treatment options to accelerate BV regression, the latter being of high clinical relevance. By contrast, targeting macrophages to facilitate a faster regression of LV might be of clinical use in treating corneal surface diseases that have been shown to be mediated by persistent corneal LV such as dry eye disease and ocular allergyAll animal experiments were approved by the local animal care committee  and were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were kept under standard pathogen-free conditions in a 12 h light-dark cycle. Prior to suture placement, suture removal, corneal incision and subconjunctival injections, mice received injection anesthesia consisting of ketamine (100 mg/kg) and xylazine (20 mg/kg). Additionally, oxybuprocaine hydrochloride eye drops  were applied as local anesthetic.56. Mice received atropine sulfate eye drops  20 minutes prior to surgery to avoid iris incarceration. To generate a perforating corneal incision with 1 mm length, the corneal center was marked with a 1.0 mm trephine. The cornea was perforated with a 30-gauge needle and a sagittal linear incision was inflicted using surgical micro scissors. After incision injury, mice received ofloxacin eye drops  three times daily during the first three days.Corneal perforating incision injury was performed in the right cornea of 6–8 week old female C57Bl/6 N mice , as previously described57. Briefly, three figure-of-eight 11-0 nylon sutures  were placed intrastromally in the cornea of the right eye of 6–8 week old female Balb/c mice . Each figure of eight-suture was placed with two stromal incursions extending over 120 degrees of the corneal circumference. Sutures were left in place for 14 days, if not indicated otherwise.The mouse model of suture-induced corneal neovascularization was performed as previously describedFor macrophage depletion mice received subconjunctival injections of 10 µl mannosylated clodronate liposomes  every other day for the duration of one week. Control mice received equal amounts of mannosylated PBS-Liposomes. To deplete corneal macrophages at different time points after suture placement and incision injury, clodronate liposomes were administered in a time restricted manner using different injection regimens Fig. . At the Mice were sacrificed at the indicated time points, and corneas were excised, rinsed in PBS, and incubated in 20 mM EDTA in PBS for 30 min at 37 °C for removal of the corneal epithelium. Subsequently, corneas were washed, fixed in ethanol, and blocked in 2% BSA. For the analysis of corneal macrophages, BV, and LV, corneas were stained with FITC-conjugated anti-CD31  and anti-LYVE-1  , or anti-F4/80  overnight. To detect LYVE-1 or F4/80 corneas were washed and incubated with Cy3 goat-anti rabbit  or Alexa fluor 555 goat anti-rat secondary antibody  for 45 min. After final washing, corneas were mounted on microscope slides with fluorescence mounting medium .57. Briefly, for the detection of bright vessels on dark background, gray-scale pictures of fluorescence stained corneal whole-mounts were modified using several software-based filters to sharpen images and increase contrast. After manually setting a threshold for detection, bright corneal vessels were detected on black background in an automatic manner. Results are expressed as the percentage of corneal area covered by BV or LV. The total corneal area was defined using the outermost corneal limbus vessel as outer limiting border . Semi-automatic morphometric image analysis was performed using Olympus CellF software  as described previouslyOn day 7 after incision or suture injury, central corneas (n = 3 per group) sparing the limbal area were excised and placed into RNA later solution  until further processing. Tissue disruption was performed using Precellys 24 tissue homogenizer . RNeasy Micro Kit  was used for total RNA isolation. Complementary DNA (cDNA) synthesis was performed in random hexamer primed reverse transcriptase reactions . Primers for real-time PCR  were designed using Primer3 software and BLAST . Primer sequences are provided in suppl. table 6 cells per group were seeded in 6-well plates and allowed to re-adhere overnight. The following day macrophages were stimulated with 100 ng/ml IFNγ and 10 ng/ml LPS  or IL-4 and IL-13 (100 ng/ml each). Cytokines were obtained from PeproTech . Control cells were left unstimulated. Total RNA from macrophages was isolated 24 h later using QIAshredder columns  for cell disruption and the Qiagen RNeasy Micro Kit . cDNA synthesis and real-time PCR were performed as described above.Bone marrow-derived macrophages were isolated from female C57Bl/6 N mice (n = 3) according to standard protocols. Cells were cultured for 7 days in RPMI medium  containing 10% FBS  in the presence of 20 ng/ml M-CSF . Afterwards, macrophages were detached from culture dishes using accutase solution  and 1 × 10Statistical analyses were performed using GraphPad Prism version 6.07 for Windows . Significance was analyzed using Student’s t-test for parametric or Mann-Whitney-U test for non-parametric data sets. One-way analysis of variance (ANOVA) was used to compare BV, LV and macrophage numbers at different time points to uninjured controls and to compare mRNA expression levels in corneal tissue. A value of p ≤ 0.05 was considered statistically significant. All data are presented as single or grouped values with mean and standard-deviation (SD).Supplementary Dataset 1


Pancreatic pseudocyst is a very common benign cystic lesion of the pancreas. It develops in 5–15% of patients with peri-pancreatic fluid collection following acute pancreatitis. Collection usually occurs within the lesser sac of the omentum (near the pancreatic head and body region). But in 20–22% cases, that may be extra-pancreatic like in the mediastinum, pleura, in the peritoneal cavity including the pelvis. The pancreatic pseudocyst typically contains brownish fluid with necrotic tissue sludge which may get infected giving rise to infected pseudocyst or pancreatic abscess. The present case is an unusual condition of a young alcoholic subject who was finally diagnosed as a case of a pancreatic abscess within hepato-gastric ligament and was managed with operative intervention.To the best of the author’s knowledge, it is the first-ever reported case of a pancreatic abscess within the hepato-gastric ligament in the world. Literature was reviewed to explore potential etiopathogenesis and therapeutic strategies of this extremely rare condition.3) with neutrophilia and elevated plasma pancreatic amylase and lipase levels. USG and MDCT scan of the whole abdomen revealed a thick-walled echogenic cystic swelling of size 18 cm × 12 cm in the epigastric region. USG guided aspiration of the cyst revealed mixed purulent brownish fluid. The cyst fluid was negative for mucin stain and contained high amylase level with low CEA level, suggesting infected pancreatic pseudocyst.A 38 years old gentleman, chronic alcoholic, having a previous history of acute pancreatitis 3 months back, presented with fever (102 degrees Fahrenheit) and a huge [20 cm  X 15 cm ] severely painful swelling in the epigastric region. The swelling was round-shaped, intra-abdominal, fixed to deeper tissue, tense-cystic, poorly trans-illuminant, non-pulsatile and irreducible. Routine blood tests showed leucocytosis (14,500/mm. Aspiration of the pus mixed cyst fluid along with tissue debris was done. Through irrigation of the cyst was done with normal saline. The cyst wall was de-roofed leaving a small part adherent to the inferior surface of the left lobe of the liver. Later the cyst fluid culture showed significant growth of Escherichia coli. He was put on IV antibiotics. The patient was discharged in a stable condition after 5 days. The histopathological examination confirmed pancreatic abscess.An open drainage procedure was considered through an upper midline laparotomySix months after the operation, the patient is doing well, remaining asymptomatic and there is no sign of recurrence.Due to extreme rarity, pancreatic abscess formation within hepato-gastric ligament may be a diagnostic dilemma and requires a high index of suspicion. Surgeons should be aware of this rare clinical entity for prompt management of potential morbidity. The pancreatic pseudocyst is a very common benign cystic lesion of the pancreas. It develops in 5–15% of patients with the peri-pancreatic fluid collection, usually 4 weeks after the initial attack of acute pancreatitis. Although collection usually occurs within the lesser sac of the omentum (near the pancreatic head and body region), in 20–22% cases, that may be extra-pancreatic . Intra-hTo the best of the author’s knowledge, it is the first-ever reported case of a pancreatic abscess within the hepato-gastric ligament in the world. Due to extreme rarity, pancreatic abscess formation within hepato-gastric ligament may be a diagnostic dilemma and requires a high index of suspicion. Literature was reviewed to explore potential etiopathogenesis and therapeutic strategies of this extremely rare condition.. The swelling, initially mild dull aching and smaller in size, started 2 months back and gradually attained the present size [20 cm  X 15 cm ] in the last 7 days. He gradually developed tenderness and induration over the swelling after a trivial blunt trauma on the area 7 days back. General examination revealed a normotensive person (BMI of 19.6) without any ascites, dysuria or any sign of intestinal obstruction. He was significant only for the swelling which was located in the epigastric region. The swelling was round-shaped, intra-abdominal, fixed to deeper tissue, tense-cystic, poorly trans-illuminant, non-pulsatile and irreducible. The swelling showed no expansile impulse on coughing and was devoid of any bowel sound within it. He neither had a history of any previous abdominal surgery nor had he received any treatment for this disease. He had no history of coagulopathy or similar illness in his family.A 38 years old gentleman, chronic alcoholic, having a previous history of a single episode of hospitalization with acute pancreatitis 3 months back, presented with fever (102 degrees Fahrenheit) and a huge severely painful swelling in the epigastric region 3;reference range 4000-8000/mm3) with neutrophilia (81%) and elevated plasma pancreatic amylase and lipase . MRCP failed to show any communication of the cystic SOL with pancreatic ducts.The possibility of pyogenic liver abscess, infected hydatid cyst, post-traumatic infected hematoma or infected pseudocyst of the pancreas was considered clinically. Urgent relevant blood tests and urine analysis were within normal limits except leucocytosis . Aspiration of the thick pus mixed cyst fluid, along with tissue debris, was done avoiding any spillage in the abdomen. Through irrigation of the cyst was done with normal saline. The cyst wall was de-roofed leaving a small part that was adherent to the inferior surface of the left lobe of the liver.  A portion of omentum was securely placed over the cleaned inner surface of the residual cyst wall.  Abdomen was closed leaving a positive pressure closed abdominal drain over the operative site. The cyst wall and tissue debris were sent for histopathological examination.After proper resuscitation and pre-operative workup of the subject, considering the presence of (on pre-operative USG Scan) high volume non-drainable tissue debris and abscess formation within the cyst, open drainage procedure was considered through an upper midline laparotomy. On entering the abdomen, one large thick-walled cyst was identified within the hepato-gastric ligament. Fig. . Aspirater. Fig.  A portioll. Fig.  Abdomen Escherichia coli. He was put on IV antibiotics according to the drug sensitivity report. Oral feeding was started the next day. He became afebrile after 2 days and early ambulation was encouraged. On the third postoperative day, after removal of the drain, total collected 110 ml of serosanguinous fluid from the abdomen was again sent for study. Post-operative serum amylase, lipase level came to be normal as well as pancreatic enzymes level in drain fluid was also found negligible. There was no surgical site infection. The patient was discharged in a stable condition after 5 days.His postoperative stay in the hospital was uneventful. The cyst fluid culture showed significant growth in  suggesting chronic infective cystic lesion. Although no feature of granuloma or malignancy could be detected.The histopathological confirmation of pancreatic abscess was made when multiple sections from the cyst wall showed features of compressed fibro-collagenous tissue infiltrated by mixed inflammatory cells . The tissue debris also showed features of necrosis with rich inflammatory infiltrates Fig. a & b sugSix months after the operation, the subject is asymptomatic and has returned to his usual life without any clinical sign of recurrence. He was advised regular follow up in our surgical Out-Patient Department (OPD) to detect any recurrence by clinical examination and ultrasonography.Pseudocyst of the pancreas is a very common cystic lesion of the pancreas which comprises 75% of all cysts of the pancreas. The other 25% are cystadenomas, IPMNs, cystic necrosis of adenocarcinoma, and congenital polycystic disease. Pancreatic pseudocyst develops in 5–15% of patients with peri-pancreatic fluid collection following acute pancreatitis. It occurs when sequestered pancreatic fluid collection localizes and fibrotic reaction completes usually 4 weeks after the initial attack of acute pancreatitis, in or around the pancreas. But 20–40% of the pseudocyst of the pancreas resolves spontaneously. Trauma and recurrent/chronic pancreatitis may cause rupture of pancreatic ducts which also gives rise to pseudocyst formation due to increased ductal pressure. The capsule of the pseudocyst is composed of collagen and granulation tissue with a lining fibrin layer which lacks epithelium [Although collection usually occurs within the lesser sac of the omentum, it can also be found in another ectopic (extra-pancreatic) sites depending on where the pancreatic enzymes were released and the path it had traveled. Most frequently it is localized within the pancreatic head and body region, but in 20–22% cases, they may be extra-pancreatic  in a locUp to 8 th weeks of intra-uterine life, ventral mesentery contains ventral anlage of the pancreas and the remnant of the dorsal portion of the ventral mesentery remains after birth as gastro-hepatic and gastro-duodenal ligaments. So, pancreatic sequestered fluids from the head of the pancreas after acute pancreatitis can spread towards porta hepatis, left lobe of the liver , 6 or evSeveral proposed mechanisms explain how pseudocyst of the pancreas occurs after acute pancreatitis in different extra-pancreatic locations. These are as follows-.2.Escherichia coli) giving rise to infected pseudocyst or pancreatic abscess.In extreme rarity (very few reported cases) pancreatic pseudocyst may be found located within the hepato-gastric ligament when ascend of pancreatic fluids get arrested within it. The pancreatic pseudocyst typically contains brownish fluid with necrotic tissue sludge. These materials may get infected commonly by gram-negative rods  , up to 5The clinical diagnosis is corroborated by MDCT or MRCP which is very helpful especially in the case of extra-pancreatic locations. MRCP, in addition to information given by MDCT like cyst-wall thickness and nature of contained fluid (hemorrhagic/infective), can show communication with other organs. The presence of gas bubbles may be seen in the case of abscess formation on radiological assessment. Endoscopic ultrasound (EUS) with FNAC may be needed in whom the diagnosis is unclear, as rarely pseudocyst may form due to underlying malignancy. Characteristics features of pancreatic pseudocyst aspirate include high amylase level but low CEA level and absent mucin. Serum amylase level, ten times greater than normal, has 98% specificity and 73% sensitivity for detecting pseudocyst of pancreas. Observation is indicated for asymptomatic patients with cyst diameter lesser than 4 cm, located in the pancreatic tail region without any duct obstruction/communication as they may undergo spontaneous resolution . PseudocInvasive therapies are indicated for symptomatic patients and when the possibility of a cystic neoplasm can’t be ruled out. When most of the pancreatic pseudocyst, if needed, is treated with decompressive procedures rather than resection, pancreatic abscess in ectopic sites like within hepato-gastric ligament should be resected/de-roofed after proper drainage (avoiding spillage) and thorough irrigation.There is increasing evidence that transduodenal/transgastric endoscopic drainage of non-infected thick-walled pseudocyst is safe and effective modality when it is located in close contact (< 1 cm) with duodenum or stomach respectively . There sSurgical (open/laparoscopic) intervention is needed for failed or unsuitable endoscopic cases. Cystogastrostomy or cystoduodenostomy should be done, as internal drainage procedures, where the cyst is located near the stomach or duodenum respectively. The cysts, which are not in contact with those two organs, need Roux-en –Y cystojejunostomy.Surgical cysto-enterostomy is highly successful in achieving immediate drainage in more than 90% cases with 12% chances of recurrence in the long term depending upon location and etiology. Although this procedure carries 1–5% mortality and 10–30% morbidity .Percutaneous external drainage has a high incidence of external fistula formation, so it may be tried in septic (high-risk group) patients secondary to pseudocyst infection/abscess only. Percutaneous drainage may also be tried in case of an unruptured, noninfected cyst with the intact pancreatic duct. While simple aspiration is associated with a 70% recurrence rate, placement of percutaneous drainage catheter provides a 90% recurrence-free resolution . AnotherTo the best of the author’s knowledge, it is the first-ever reported case of a pancreatic abscess within the hepato-gastric ligament in the world.Despite the extreme rarity, pancreatic abscess formation within hepato-gastric ligament may be a diagnostic dilemma and requires a high index of suspicion. Patients with a pancreatic abscess in hepato-gastric ligament may present with toxic features and epigastric lump. After proper resuscitation and confirmation of the disease, the cyst should be resected/de-roofed after proper drainage (avoiding spillage) and thorough irrigation. Proper antibiotics, according to culture and sensitivity test of cyst fluid, should be administered. Surgeons should be aware of this clinical entity during the management of suspected pathology involving the epigastric region for prompt management of potential morbidity.


Here, we test the extent to which forest management has driven fire regime shifts in a temperate forest landscape. We combine multiple fire proxies  derived from highly resolved lake sediments , and apply a new statistical approach to classify source area- and temperature-specific fire regimes . We compare these records with independent climate and vegetation reconstructions. We find two prominent fire regime shifts during the 19th and 20th centuries, driven by an adaptive socio-ecological cycle in human forest management. Although individual fire episodes were triggered mainly by arson  during dry summers, the biomass burnt increased unintentionally during the mid-19th century due to the plantation of flammable, fast-growing pine tree monocultures needed for industrialization. State forest management reacted with active fire management and suppression during the 20th century. However, pine cover has been increasing since the 1990s and climate projections predict increasingly dry conditions, suggesting a renewed need for adaptations to reduce the increasing fire risk.Fire regime shifts are driven by climate and natural vegetation changes, but can be strongly affected by human land management. Yet, it is poorly known how humans have influenced fire regimes prior to active wildfire suppression. Among the last 250 years, the human contribution to the global increase in fire occurrence during the mid-19


The development of ‘molecular-omic’ tools and computing analysis platforms have greatly enhanced our ability to assess the impacts of agricultural practices and crop management protocols on soil microbial diversity. However, biotic factors are rarely factored into agricultural management models. Today it is possible to identify specific microbiomes and define biotic components that contribute to soil quality. We assessed the bacterial diversity of soils in 51 potato production plots. We describe a strategy for identifying a potato-crop-productivity bacterial species balance index based on amplicon sequence variants. We observed a significant impact of soil texture balances on potato yields; however, the Shannon and Chao1 richness indices and Pielou’s evenness index poorly correlated with these yields. Nonetheless, we were able to estimate the portion of the total bacterial microbiome related to potato yield using an integrated species balances index derived from the elements of the bacterial microbiome that positively or negatively correlate with residual potato yields. This innovative strategy based on a microbiome selection procedure greatly enhances our ability to interpret the impact of agricultural practices and cropping system management choices on microbial diversity and potato yield. This strategy provides an additional tool that will aid growers and the broader agricultural sector in their decision-making processes concerning the soil quality and crop productivity. Soil microbial communities are impacted by meteorology ,2, soil soil quality as “the capacity of a soil to function within ecosystem and land-use boundaries to sustain biological productivity, maintain environmental quality, and promote plant and animal health”. According to Pankhurst et al., (1997) , us-Clune 16 [20] cIn agro-ecosystems, soil quality is assessed to enhance and sustain productivity. As for natural ecosystems, soil quality is monitored to favor the maintenance of environmental quality and biodiversity conservation . A necesHistorically, soil quality is measured by monitoring abiotic, biochemical and biological indicators. Abiotic indicators include soil texture, wet aggregate stability, available water capacity, soil hardness, pH, cation exchange capacity and extractable nutrients. Biochemical indicators include organic matter, active carbon and the soil protein index. Finally, biological indicators include microbial respiration rate, nitrogen mineralization potential and microbial biomass –24. ThesThe lack of detailed information hinders the development of efficient land-use management strategies that could address the benefits humans derive from ecosystem services and the advantages and trade-offs derived from soil-based ecosystem services. The links between soil biota diversity/composition, soil functions and soil-based ecosystem services have been well established ,26–29. MMicrobial communities play key roles in ecosystem processes by driving the Earth’s biogeochemical cycles and diversity metrics related to the richness, evenness, and phylogenetic diversity of soil microbial communities. They can be used to analyze the functional consequences of variations in soil microbial diversity ,30. FuncAmong microbial taxonomic groups, some sharing similar functionality are influenced by agricultural practices. This suggests that, cropping practices may allow manipulation of influential community members . Other iThe taxonomic composition of potato rhizosphere bacteria at various stages of plant development was stable in the face of very diverse environmental conditions . An ‘oppThese recent studies spurred us to undertake this investigation, which has the following objectives: i) determine the physico-chemical, agronomic, climatic characteristics prevailing for each georeferenced soil sampled in 51 potato field plots; ii) determine the soil bacterial alpha- and beta-diversity of each soil sample; iii) apply an innovative selection procedure using physico-chemical, climatic and biological indicators to select amplicon sequence variants that correlate with potato yields; and iv) develop an integrative approach using a species balance index linked to potato productivity. The project’s paramount goal is to provide a state-of the-art tool that will assist agricultural stakeholders in their decision-making processes concerning the quality of soil and crop productivity.This study was carried out on private agricultural lands. The owners of the agricultural lands are collaborators in our research project. They gave us permission to conduct this study on their lands.2 area in the field for a total of 4 samples per field. Each soil sample consisted of four soil cores (6 cm x 20 cm) taken on the row between two potato plants and each core was sampled at a corner of a 1m2 quadrant. The four soils cores of each sample were manually homogenized, and the soil samples were placed in sealed bags and kept on ice before being quickly stored at -80°C prior to DNA extraction. A summary of the samples is presented in During the 2013 and 2014 potato flowering period, 6 cm x 20 cm soil cores were collected from 51 geo-localized (GPS coordinates) sampling plots in the Province of Quebec in Canada. The plots were located in 13 potato fields. Each experimental plot was located at a corner of a 2500 m2 solution (1:1 v/v). Soil texture was determined by sedimentation ) of a D-parts composition, rk and sk are the numbers of components in the respective right and left subsets of the kth balance. Parts x with subscript i belong to the right subset in the numerator and parts x with subscript j belong to the left subset in the denominator. The coefficient Ever since John Aitchison provided solutions to spurious statistics using compositional data , the trailr)  to generilr)  and compilr value is positive and conventionally placed to the right of the axis.As in clr) ASVs done with the feature-table plugin implemented in QIIME 2.Recent research –55 stresclrs  balance, pH, and weather were observed.residual yield, are extracted they can be related to the biological components of the soils.Once the residuals of the regression, here referred to as the clrs of ASV counts with residual potato yields. From these correlations, we retained ASVs associated to significant correlations at the 0.05 level. We identified 79 positively correlated and 61 negatively correlated ASVs from a total of 2008.We correlated the The Verrucomicrobia, Armatimonadetes and other less abundant phyla were absent from the retained ASVs. The most of retained ASVs were from the Proteobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, Gemmatimonadetes and Acidobacteria phyla. In the case of Proteobacteria, Bacteroidetes and Acidobacteria, there was a higher number of retained ASVs that positively correlated with residual potato yield. Among the the Saccharibacteria and Nitrospirae phyla, we observed no clrs of ASVs counts that negatively correlated with the residual potato yield. This suggests that among these six bacterial phyla, most retained ASVs contribute positively to potato crop productivity.Retained ASVs belong to 12 bacterial phyla. The total and retained abundance patterns of ASV were similar, although species balance index related to potato yield (SBI-py) was computed as the log-ratio between the ASV counts associated to positive (numerator) and negative (denominator) significant correlations (at the 0.05 level) between ASV clrs and potato residual yields. A The three alpha diversity indices were barely correlated with potato yield and residual potato yield. The Pielou index correlated less well with potato productivity than did the other two alpha diversity indices. Alpha diversity indicators have been used to assess impact of land uses  and of dclrs of ASV counts that correlated positively or negatively with this residual yield have been used to determine a species balance index related to potato yield (SBI-py). This innovative selection procedure of ASVs lead by design to good correlations between the newly developed species balance index (SBI-py) and the residual potato yield of the field plots sampled.It has been reported that specific guilds of taxa among the soil bacterial microbiome can be selected to modify plant traits and to coordinate changes in soil resource pools ,35,36. TBurns et al.,  reportedAlso, a detailed statistical analysis on the operational taxonomic unit (OTU) level, representing bacterial species detected in potato rhizosphere, revealed a stable component and a dynamic component of a core microbiome related to potato crop and field sites sampled . As obseThe use of ASV instead of OTU tables or taxonomic tables allowed a better appreciation of the complexity and species variability among soil microbiomes. The accuracy of the taxonomic tables  is limited due to the reference databases that are used for each OTU or ASV taxonomic assignations. This limitation is even more important with the use of OTUs made with 97% similarity between sequences instead of ASVs.clrs of ASVs. The positively and negatively significantly (at the 0.05 level) correlated clr-transformed ASV counts at each site (greyish dots) are plotted along the contours of the species balance index scores. By design, the SBI-py contours follow the pattern of positive and negative ASVs.The triplot shown in The obvious delineation of positive and negative ASVs zones indicate potential ecological niches related to favorable and unfavorable soil microbiomes for potato cropping systems. Because the  balance vector points in an opposite direction of the high SBI-py positive values, we infer that soils with higher clay proportions are linked to higher SBI-py positive values. The same reasoning applies to the [N | C] balance: higher nitrogen to carbon content is linked to higher SBI-py positive values. On the other hand, high organic contents,  and sandy soils  are linked to negative SBI-py index values. The Shannon diversity index of rainfalls [SDI] points through a negative SBI-py values, indicating that a more even distribution of precipitations is linked to low SBI-py values. Finally, although the pH vector does not follow the highest SBI-py gradient, we can find relatively high SBI-py values at low pH.Our results highlight the importance of using soil bacterial composition as a biological index of soil quality and, more specifically, of crop productivity. We developed the SBI-py index based on the evidence that specific components of the soil microbiome can explain aspects of potato productivity. While, by design, the SBI-py showed a high correlation with yield detrended from several environmental conditions (0.77), three commonly used alpha-diversity indices  poorly correlate to potato productivity. The approach we employed to develop the SBI-py index could be further validated using a larger sample size combined with machine learning techniques.The soil microbiome encompasses different prokaryotic and eukaryotic organisms dispersed along a complex interacting food web, thus providing numerous soil ecosystemic functions that may promote plant growth . For exaWe have recently undertaken a study of the interactions between bacterial and fungal diversity in soil and rhizosphere of potato cropping systems to evaluate their impact on potato yield and soil productivity.Our results represent a significant contribution to research aimed at selecting biotic data that give more detailed information on soil biota than the usual biological indicators. These biotic data can be incorporated as biological indicators into the determination of soil quality indices. Our strategy for the selection of significant ASVs in specific component of soil microbiome can also be implemented in a variety of agricultural applications.


Observational studies are suited to examining links between the routinehospital management of self-harm and future suicide and all-cause mortalitydue to their large scale. However, care must be taken when attempting toinfer causal associations in non-experimental settings.Data from the Multicentre Study of Self-Harm in England were used to examineassociations between four types of hospital management  following self-harm and risks of suicideand all-cause mortality in the subsequent 12 months. Missing data werehandled by multiple imputation and propensity score (PS) methods were usedto address observed differences between patients at baseline. Unadjusted, PSstratified and PS matched risk ratios (RRs) were calculated.The PSs balanced the majority of baseline differences between treatmentgroups. Unadjusted RRs showed that all four treatment types were associatedwith either increased risks or no change in risks of suicide and all-causemortality within a year. None of the four types of hospital management wereassociated with lowered risks of suicide or all-cause mortality followingpropensity score stratification  and propensity score matching , though there was no longer an increased riskamong people admitted to a psychiatric bed. Individuals who self-cut were atan increased risk of death from any cause following psychosocial assessmentand medical admission. Medical admission appeared to be associated withreduced risk of suicide in individuals already receiving outpatient or GPtreatment for a psychiatric disorder.More intensive forms of hospital management following self-harm appeared tobe appropriately allocated to individuals with highest risks of suicide andall-cause mortality. PS adjustment appeared to attenuate only some of theobserved increased risks, suggesting that either differences betweentreatment groups remained, or that some treatments had little impact onreducing subsequent suicide or all-cause mortality risk. These findings arein contrast to some previous studies that have suggested psychosocialassessment by a mental health specialist reduces risk of repeat self-harm.Future observational self-harm studies should consider increasing the numberof potential confounding variables collected. Preventing suicide is a global public health priority. Annually there are 11.4suicides per 100,000 population and it is the leading cause of death for 15 to29-year olds . While sThe treatment provided to individuals presenting to ED following self-harm can varywidely . RecommeSome studies have attempted to identify possible protective approaches to carefollowing a hospital attendance. A randomised controlled trial (RCT) carried out inEngland provided therapeutic assessments to adolescents following an ED attendancefor self-harm . TheintFindings from observational studies, such as case-control and cohort studies, arebetter powered to measure suicide and other causes of mortality as outcomes. Someobservational studies have suggested increased risks of mortality following hospitalcare. A case-control study carried out in Denmark reported increased risks ofsuicide in the year following emergency department treatment and psychiatricadmission . The incInferring causality between treatment and an outcome is a major challenge withobservational data. In randomised trials the risks of the outcome of interestfollowing treatment would be expected to be equal if the treatment groups wereswitched (known as exchangeability) . SubsequPrevious studies have employed propensity score methods to compare population-leveltreatments following self-harm , 24. HowThe average treatment effect (ATE) or the average treatment effect on the treated(ATT) can be estimated. If both treatment groups are represented across the fullrange of PSs, it is appropriate to estimate the ATE. If one or more of the treatmentgroups has a limited range of PSs, for example if a treatment was only given toindividuals with high PSs and there were no treated individuals with lower PSs, anestimate of the ATT is more appropriate . The ATEThe aim of the current study is to use propensity score methods to estimate theeffects of four categories of care received after self-harm  on suicide and all-cause mortality. We hypothesised that,following propensity score adjustment, individuals receiving each category ofmanagement would be at lower risk of suicide and all-cause mortality than thosereceiving no such care. In addition to overall associations, we were interested indifferences between certain groups of individuals. There is evidence that effects ofroutine management of self-harm may differ by mental health care and self-harmhistory . EffectsWe used data from the Multicentre Study of Self-Harm in England , an obseWe included adults aged 16 years and over presenting between 2000 and 2010 fortwo of the centres, and from 2003 to 2010 for one centre, due to dataavailability. For the ‘admission to a medical ward’ treatment category, for oneof the centres, data were only available from 2005. Each individual’s finalepisode of non-fatal self-harm, either before death, or within the study period,was included. The cohort included 31,725 individuals attending one of the studyhospitals between 2000 and 2010. 42.1%  presented to Manchester, 30.5% to Derby and 27.4%  to Oxford. Individuals were followed up untilthe end of 2012 and notifications of deaths were provided by the Data LinkageService, part of the Health and Social Care Information Service [The self-harm monitoring system in Oxford was approved by South Central–BerkshireNational Research Ethics Service and Derbyshire Research Ethics Committeeapproved the study in Derby. Both were granted ethical approval to collect datafor both local andmulticentre projects. South Manchester Research Ethics Committee reviewed theproject inManchester. The project was deemed not to require approval as the monitoring isconducted as part of a clinical audit system. All centres have approval underSection 251 of the NHS Act (2006) to collect patient identifiable data withoutpatient consent and Oxford, Derby and Manchester have consent to send patientdetails to the Data Linkage Service.To enable us to examine effects of each management strategy separately,treatments were grouped into four main categories: psychosocial assessment by amental health specialist, admission to a medical ward , referral to psychiatric outpatient services and admission to a psychiatric bed.Treatment categories were not mutually exclusive. For example, individuals couldreceive a specialist psychosocial assessment and then be referred to outpatientcare.Effects of each treatment category were estimated separately. The comparisongroups were individuals not receiving the treatment of interest, combined into asingle comparison group, regardless of other types of management allocatedduring their presentation. Propensity score methods  helped to ensure that individuals in the comparison groupswere similar to those in the treatment groups, in terms of their measuredcharacteristics. For individuals referred for outpatient psychiatric care, thoseadmitted to a psychiatric bed  were excluded from the comparisongroup. Due to the acute and high-intensity nature of psychiatric admission, anyeffect is likely to obscure that of outpatient care. Furthermore, people whowere admitted as a psychiatric inpatient would be likely to have receivedoutpatient care after discharge .The outcomes of interest were suicide and death from any cause within twelvemonths of the hospital presentation. Suicides were defined as deaths given ICD10 codes of intentional self-harm (X60 to X84) or undetermined intent (Y10 toY34) . All-cauBoth suicide and all-cause mortality within the twelve months following an EDpresentation for self-harm are relatively rare events , 33. AssMissing data were handled in one of two ways. Variables with low proportionsof missing data (<6%) included the four categories of hospitalmanagement, suicide and all-cause death, age, gender and IMD area-leveldeprivation score. These data were found to be missing at random and caseswith any missing values were excluded, an acceptable approach when thepercentage of missing data is small . For varWe estimated a propensity score for each individual in the cohort. Apropensity score represents the probability of treatment assignment based onobserved characteristics . We usedIn order to determine the most appropriate propensity score approach, the‘common support’  was examined. Therewas good common support across all propensity score values for psychosocialassessment and medical admission Figs. ThThere was common support only for propensity scores up to 0.6 for individualsreferred to outpatient mental health care; above this threshold there werefew untreated individuals (n = 785) . SimilarUnadjusted risk ratios (RRs) between treatment groups within 12 months werecalculated separately for suicide and all-cause mortality using log-binomialregression models in Stata (Version 13.1) . For tre18,252/31,725 (57.5%) individuals received an assessment by a mental healthspecialist, 15,738/25,270 (62.3%) were admitted to a medical bed, 9,244/29,889(30.9%) were referred to outpatient psychiatric care and 1,800/31,725 (5.7%)were admitted to a psychiatric bed. There were baseline differences between the treatment groups . PS adju217 (0.68%) individuals died by suicide in the 12 months following the self-harmepisode. The rate of suicide was higher amongst individuals receiving each ofthe four categories of management, rising most sharply for individuals receivinga referral to outpatient psychiatric care  and thoseadmitted to a psychiatric bed  . FollowiThe proportion of individuals dying from any cause within 12 months of self-harmwas 2.17% (687). There was no change in risk of death following specialistpsychosocial assessment, prior to any PS adjustment. Individuals admitted to amedical bed, referred for outpatient psychiatric care or receiving psychiatricadmission were at greater risk of death  compared to those whoself-poisoned  or used a method of harm other thanpoisoning or cutting .The increased risk of suicide following medical admission was even higher forfemales  and those who harmedthemselves using a method other than poisoning or cutting . For individuals in treatment for a psychiatric disorderat the time of the self-harm, medical admission was associated with adecrease in the risk of 12-month suicide .Individuals who used cutting to harm themselves were at higher risk of deathfrom any cause .The increased risk of all-cause mortality following referral to outpatientpsychiatric care was more pronounced for adults aged between 45 and 64  than other age groups. For individualswith a history of self-harm in the past year, the increased risk ofall-cause mortality associated with psychiatric outpatient referral was notseen .The risk of all-cause death within 12 months of psychiatric admission waslower amongst those who had self-harmed within the past year .Prior to adjustment, the receipt of any of the categories of hospital managementwas associated with either an increase or no change in risks of suicide andall-cause mortality. None of the four types of hospital management wereassociated with lowered risks of suicide or all-cause mortality followingpropensity score adjustment (either by stratification or matching methods),though there was no longer an increased risk among people admitted to apsychiatric bed.Some interactions between subgroups and estimated treatment effects were found.Individuals who self-cut were at an increased risk of death from any causefollowing psychosocial assessment and medical admission. Medical admissionappeared to be associated with reduced risk of suicide in individuals alreadyreceiving outpatient or GP treatment for a psychiatric disorder.This study has made use of a large, population-level cohort, enabling suicide andmortality  to be compared betweentreatment groups. The routine nature of the treatments means studying theireffects in an experimental setting is difficult. The use of propensity scoremethods to address observed sources of selection bias is a rigorous approach inthis setting. However, the approach does have limitations. There is likely to beremaining imbalance in unobserved characteristics between treatment groups. Anyobserved links between treatments and outcomes cannot be assumed to be causaland should be interpreted with this caveat. The associations may be due,instead, to unmeasured baseline differences between individuals who receiveddifferent treatment. There were also some measured differences between treatmentgroups after PS adjustment. Although the numbers of individuals receiving mosttreatment types were adequately powered to detect differences in suicide ratesbetween treatment groups, the group receiving psychiatric inpatient admissionwas relatively small. This limited the statistical power for this estimate oftreatment effect, particularly for detecting smaller differences.This study examined broad categories of hospital management. For example, areferral to outpatient mental health care could involve community mental healthcare, crisis resolution teams, drug and alcohol teams or psychological therapy.It was not possible to examine effects of specific aspects of treatment.Furthermore it was not possible to ascertain if individuals received the offersof follow-up care as intended, and if they did, the length of treatmentreceived. Future study designs could focus on linking hospital data tooutpatient and primary care data to examine these questions in greaterdetail.In unadjusted analyses, most aspects of hospital care following self-harm wereassociated with increased risks of suicide and all-cause mortality. This is inline with research from other countries, and suggests services are appropriatelyidentifying treatment needs. Following propensity score methods to addressobserved confounding factors, risks following specialist psychosocial assessmentand psychiatric admission were attenuated, suggesting some of the selection biaswas addressed by the PS methods. It is likely that there were importantunmeasured confounding factors that, had we been able to account for, may havealtered our results.The increased risks of suicide and all-cause mortality observed followingreferral to outpatient care could reflect incomplete PS balancing , residual unobserved confoundingor risks associated with treatment. The findings may reflect shortcomings in theaftercare provided for people referred to outpatient mental health services, thelack of evidence for effective interventions or they may result from incompleteadjustment for confounding factors.The results from this study differed from an observational study conducted usingJapanese general hospital data . PatientA recent systematic review of RCTs of psychosocial treatments for adults who hadself-harmed found evidence  thatcognitive-behavioural-based psychotherapy could help prevent repeat self-harm. HoweverIncreased risks following medical admission and referral to psychiatricoutpatient care were not seen for individuals with a history of self-harm andpsychiatric treatment. The reduced risk of suicide following medical admissionfor these individuals could reflect benefits of spending time in hospital withmore time for existing follow-up support to be arranged. The self-harmpresentation may have helped to trigger an increase in treatment intensity,helping to re-establish contact with sources of care. This could have providedsome protection against future self-harm and mortality for the year ahead, whichwas not received by those who were not known to services beforehand. However,the level of care received before the referral following self-harm, in terms oftreatment intensity and setting (e.g. outpatient or primary care), was not knownfor individuals in this study.Expanding the scope of the variables included in self-harm cohorts may help toaddress potential hidden confounding when comparing patient outcomes. Asystematic review of risk factors for repeat self-harm found that the presenceof depressive symptoms was a key risk factor . HoweverMost aspects of hospital management following self-harm were allocated to thosewith subsequent increased risks of suicide and all-cause mortality, suggestingindividuals most at risk were receiving them. While some of these risks wereattenuated following PS methods, there were no aspects of hospital managementthat were associated with lower risks of subsequent death. Results from thisstudy may reflect incomplete adjustment for confounding effects. Moreinformation on potential confounding factors may alter our estimates treatmenteffect. In addition, more detailed information about the specific approach usedas part of an individual’s routine clinical care following self-harm may help toelucidate the relationship between hospital management and mortality risks.Future studies may be able to build further on the evidence presented here byobtaining more detailed information about the content of the routine clinicalmanagement following self-harm. Some of these factors are routinely assessed aspart of the specialist psychosocial assessment, increasing the feasibility ofincluding them in future cohort studies.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.


Dental implant diseases, peri-implantitis (PI) and peri-implant mucositis (PIM), have shown wide prevalence in recent studies. Despite the prevalence, diagnosing peri-implant disease (PID) remains challenging as common diagnostic methods of periodontal probing and radiographs may be inaccurate. These methods only document pre-existing destruction rather than current disease activity. Furthermore, there is no current model to predict the progression of PID. Though a predictive model is lacking, biomarkers may offer some potential. Biomarkers are commonly used in medicine to objectively determine disease state, or responses to a therapeutic intervention. Gingival crevicular fluid (GCF) biomarkers have moderate diagnostic validity in periodontitis. Biomarkers in peri-implant crevicular fluid (PICF) also show promising results in regard to their diagnostic and prognostic value. The aim of this review is to summarize the current knowledge of PICF biomarkers in the diagnosis of PID and evaluate their validity to predict disease progression. This review found that PICF studies utilize different methods of sampling and interpretation with varying validity (sensitivity and specificity). A number of promising diagnostic techniques were identified. Commercially available chair-side tests for MMP-8 to diagnose periodontal disease and PID activity are now available. Future directions include proteomics and metabolomics for accurate, site-specific diagnosis and prediction of PID progression. Although more research is needed, this review concludes that the assessment of proinflammatory cytokines  in the PICF may be of value to diagnose PI and PIM but current research remains insufficient to indicate whether biomarkers predict peri-implant disease progression. Teeth may be lost for several reasons and surgically placed dental implants are one replacement option . There hAs implants are becoming more common, associated disease prevalence shows a positive correlation. Peri-implant mucositis (PIM) is defined as an inflammatory lesion of the soft tissues surrounding an endosseous implant without loss of supporting bone or continuing marginal bone loss . On the There is a wide heterogeneity in defining peri-implantitis. Clinicians seek to differentiate how much radiographic bone loss would be indicative of disease, as opposed to the expected post-placement bone remodeling. The radiographic criterion for peri-implantitis is defined as bone loss of ≥3 mm according to the 2017 World Workshop on the Classification of Periodontal and Peri-implant Diseases and Conditions [Progression of PI was found to have an annual rate of bone loss of about 0.4 mm but in a non-linear and accelerating pattern . Peri-imDestructive periodontal diseases are the result of environmental, host, and bacterial factors .Similarly, PI exhibits a chronic inflammatory response to the bacterial biofilm on the tooth/implant surface . PIM prePorphyromonas gingivalis and Tannerella forsythia [Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis and Tannerella forsythia [No one specific or unique bacteria has been identified in patients with peri-implant disease (PID). When compared with healthy implant sites, PI was associated with higher counts of 19 bacterial species, including orsythia . When coorsythia . Some inorsythia . Gram-neorsythia . Howeverorsythia . NotablyThe traditional clinical method to assess implant health includes a periodontal probe to measure the pocket depths and to observe bleeding upon probing. Unfortunately, this simple tool has limitations. The absence of a periodontal ligament around implants and the prosthetic design may make assessment of pocket probing depth measurements difficult to perform and interpret. Additionally, the implant mucosal seal may have less resistance to probing compared to natural teeth. This may lead to mechanically induced bleeding when probing around healthy implants. However, the healing of the epithelial attachment seems to be complete five days after clinical probing, hence, does not seem to jeopardize the longevity of implants according to an animal study . Radiogrblood was successfully used to screen for diabetes control in a dental office setting [Early diagnosis of PID and its rate of progression are a great challenge. Assessment of biomarkers may aid in early detection of PI. Biomarkers may assist both in staging and grading of periodontitis in the case definition system of periodontitis . Peri-im setting . The aimA literature search was performed in the Pub-Med database of the US National Library of Medicine, for articles published up to October 2019 using Medical Subject Heading search terms + free text terms and in different combinations. To be included in the review, studies had to (i) be written in the English language, (ii) be published in an international peer-reviewed journal, and (iii) be on humans, while animal or in-vitro studies were supplemented by an additional search to find additional relevant supporting data. The search yields were transferred to Endnote™, version 9 . Citation tracking was completed for all identified studies included in the refined library. No restriction was placed on the year of publication for the included reports. Keywords and abbreviations used in this narrative review are shown in GCF is a physiological fluid and an inflammatory exudate originating from the gingival plexus of blood vessels in the gingival corium, subjacent to the epithelium lining of the dentogingival space. GCF flows through the external basement membrane and the junctional epithelium to reach the gingival crevice. The composition of GCF can potentially be used to detect subclinical alterations in tissue metabolism, inflammatory-cell recruitment and connective tissue remodeling . CytokinThe gingival sulcus forms a unique ecological niche for microbial colonization. As the salivary film transitions at the crown to the gingival sulcus, its composition changes and the proportion of serum proteins increases due to the proximity with the crevicular fluid. The microbiota of the dental plaque biofilm drives the inflammatory process. The microbial biofilm in the gingival sulcus elicits inflammation in the surrounding connective tissue . The imbPI and periodontitis lesions exhibit critical histopathologic differences, which contribute to the understanding of dissimilarities in onset and progression between the two diseases . HistoloA variety of methods have been used to sample and analyze components of PICF targeting the diagnosis of PI. The most common method used to collect PICF has been paper strips inserted into the crevice for typically 30 s. PICF is then absorbed onto the strips. After elution from the strips into a buffer or diluent, the fluid is evaluated utilizing biomarker-specific assays. Studies have also used paper cones, membranes and microcapillary pipettes to collect PICF.The PICF volume is dependent upon the level of inflammation and pocket depth. The quantity of components collected in a deep and inflamed pocket would in all likelihood be higher than in healthy sulci. The concentration versus the quantity of the collected PICF components may offer more value in the search of valid biomarkers; however, this may be controversial . Enzyme-Most PI biomarker studies analyzed the correlation of enzymes and cytokines between healthy and diseased implants. Such components include proinflammatory cytokines such as TNFα, IL-1β, IL-6, IL-12, IL-17 and anti-inflammatory cytokines such as IL-4 and IL-10. Other cytokines analyzed were interleukin-1 receptor antagonist (IL-1ra), Granulocyte-macrophage colony-stimulating factor (GM-CSF) and salivary IL-1β. Some studies investigated chemokines such as IL-8 and macrophage inflammatory protein-1α (MIP-1α). Other reported investigations analyzed bone metabolism markers such as Receptor activator nuclear factor kappa-B ligand (RANKL), Receptor activator nuclear factor κ B (RANK), Osteoprotegerin (OPG) and Osteocalcin. Other trials investigated enzymes such as matrix metalloproteinases , tissue inhibitor of metalloproteinases 1&2 (TIMP), Myeloperoxidase (MPO), Elastase, tissue plasminogen activator (tPA), tartrate-resistant acid phosphatase (TRAP), cathepsin K (CatK). Some authors reported on other PICF components such as plasminogen activator inhibitor 2 (PAI-2), vascular endothelial growth factor (VEGF), prostaglandin E2, KLIKK-protease genes and miropin.Most investigations including systematic reviews and meta-analyses focused on the assessment of proinflammatory cytokines IL-1β and TNFα levels, demonstrating that PI sites were associated with a significant increase in their levels compared to healthy implants. IL-1β and TNFα are the two most important cytokines in osteoclast formation and bone resorption. IL-1β is mainly produced in macrophages and regulates the degradation of extracellular matrix components of the plasminogen system and collagenase activity in inflammation and wound healing. It has been shown that inhibition of IL-1β reduces tissue breakdown and the progression of tissue inflammation . TNFα inIL-10 in PICF has been shown to be negatively correlated with peri-implant disease ,38. IL-4Since RANKL and OPG are key factors regulating bone metabolism, it is likely they are involved in alveolar bone destruction in PI . HoweverHowever, one study demonstrated the presence of OPG in 79% of the PICF samples and showed a significant positive correlation between BOP, while sRANKL was only detected in 12% of PICF samples and did not show any positive correlation with clinical inflammation . AnotherAn interventional trial assessed the effects of mechanical anti-infective therapy on the levels of TNFα and OPG/RANKL ratio in healthy implant and PI sites . The resCertain enzymes in PICF, such as cathepsin K and MMPs, were heavily investigated. Cathepsin-K (CatK) is a cysteine protease that is highly expressed by osteoclasts. Its main function is hydrolyzing the extracellular bone matrix proteins. CatK was shown to be elevated in GCF from chronic periodontitis sites compared to healthy sites. CatK is a known marker of bone turnover due to its key role in remodeling and cartilage breakdown in bone by hydrolyzing extracellular bone matrix proteins. CatK is highly and quite selectively expressed in active, resorbing osteoclasts . This suMMP upregulation has been associated with irreversible peri-implant connective tissue destruction . One sugT. denticola and Prevotella intermedia showed diagnostic validation for PI in this study. IL-1β demonstrated the most significant ability for the prediction of PI disease status . The authors concluded that a combination of the above markers and microbial profiles may offer site-specific diagnosis of PID due to the increased sensitivity and specificity compared to individual biomarkers [Another investigation reported a positive correlation between MMP-8, PI, and BOP in both GCF and PICF . The automarkers . A summaIf a definitive diagnosis for PI or PIM can be made using a test with high validity, then a reasonable question would be “is the test feasible and can the test can predict progressive PI?”. Clinicians seek an easy, accurate, inexpensive and time effective test. Most diagnostic tests in dentistry are performed in a clinical setting at the dental chair. A chair-side test for the diagnosis of PIM would also be valuable if it can predict the risk of disease progression at an implant site.® [® device may be useful in differentiating active from inactive periodontal and peri-implant sites easily, quickly and inexpensively with high sensitivity and specificity (sensitivity of 90% and a specificity of 70%–85%) [A recent report presented a diagnostic chewing-gum test which could detect elevated levels of MMP-8, which was diagnostic of PI in individuals . The pat® . This is70%–85%) . Chair-s70%–85%) . This abThe conflicting results of the above studies may be due to differences in study design, material and methods utilized, such as sample collection, processing and assay sensitivity. Meta-analyses in PICF biomarkers are scarce due to heterogeneity between studies in PI diagnosis criteria. The 2017 World Workshop hopes to utilize their PI definition criteria in future clinical and research settings [The majority of PICF studies assessed only a few cytokines, enzymes or pathogens to correlate them with peri-implant diseases. Most studies lacked data on sensitivities and specificities to biomarkers in PICF; hence, the probability of false positive or false negative results could not be calculated. PICF biomarkers analyzed individually have shown mixed results or low sensitivity and high specificity values, which may weaken the biomarker’s disease predictive value. On the other hand, biomarkers of periodontal disease progression in GCF alone  provided low sensitivity and high specificity values of 23% and 95%, respectively . It is nSome reports failed to discuss important data, such as the general periodontal health in their subjects, smoking habits, systemic confounders, and other related criteria which may influence the levels of cytokines in PICF. Smoking may be an important confounder because it may affect the PICF volume and cytokine levels. Only a few studies excluded smokers. Other potential confounders may include a history of periodontitis and gingival phenotype. A retrospective study that analyzed risk factors for PID identified three predictors for PIM: history of treated periodontitis, absence of regular supportive peri-implant maintenance, and use of a bone graft . The samStandardization of PICF sampling is relevant due to the atypical morphology of the implant prosthesis. The insertion of different paper strips, cones, membranes or other devices would be technique sensitive and may offer misleading results. Biomarker concentration in the collected PICF may be adjusted for the collected volume due to the higher volume in inflamed tissues. This controversial confounder was rarely discussed in PICF studies.Biomarkers are not necessarily present in a single moment of PICF collection due to several systemic or local factors. Most studies were cross-sectional which leads to another limitation. Due to a cyclic progression of peri-implant diseases, the immune-inflammatory event biomarkers responsible for tissue breakdown may not always be active in cross-sectional studies with a single moment of fluid collection . Thus, sHost susceptibility is a critical determinant in periodontal disease pathogenesis. There was little support in the literature for a specific genotype or phenotype of immune reactivity that could be reliably used as an indicator of susceptibility to peri-implant disease. While there are varying reports of associations of specific genotypes with peri-implantitis, the studies are inconsistent . In certThe protein composition of GCF may reflect the pathophysiology of periodontal diseases. Protein profiles of GCF obtained from healthy individuals may potentially serve as a reference for identification of biomarkers of periodontal diseases by proteome analyses . The samMetabolomic analysis  that measure small degradation molecules associated with host and bacterial metabolism show promise . MetabolIt is important to mention that this review has some limitations. The outcomes from this review are limited by the heterogeneity between studies. The review only confirms evidence reported by other authors about biomarkers in diagnosing PID, but it does not give the necessary meta-analysis results because of the heterogeneity of included studies. This narrative review did not utilize the search and analytical methods of systematic reviews. Another methodological limitation is that the risk of bias of included studies was not defined.Diagnosing peri-implant diseases using periodontal probing and radiographs may be inaccurate and only provides a historical record of past disease rather than current disease activity. Developing biomarker technologies may offer possibilities in diagnostic application. Although more research is needed, the assessment of proinflammatory cytokines  in the PICF may be of value to diagnose peri-implantitis and peri-implant mucositis but are, at this time, inappropriate to predict peri-implantitis because of the limited evidence of controlled longitudinal clinical trials. Commercially available chair-side diagnostic tests for MMP-8 to detect peri-implant diseases are promising. In addition to these biomarker tools already under study, future investigations could test a multi-biomarker approach, which has been shown to demonstrate higher validity than a single biomarker. Other promising directions include the use of untargeted metabolomics and interventional, longitudinal trials to identify a unique set of biomarkers in PICF. These trials may also be able to determine the biomarkers’ validity to diagnose peri-implantitis and predict which patients and which dental implants may be at risk of disease progression. As studies show the increasing prevalence of peri-implant diseases, increasing diagnostic and predictive accuracy may have a significant impact on dental care.