Comparative Assessment of the Proteolytic Stability and Impact of Poly-Arginine Peptides R18 and R18D on Infarct Growth and Penumbral Tissue Preservation Following Middle Cerebral Artery Occlusion in the Sprague Dawley Rat.

Poly-arginine peptides R18 and R18D have previously been demonstrated to be neuroprotective in ischaemic stroke models. Here we examined the proteolytic stability and efficacy of R18 and R18D in reducing infarct core growth and preserving the ischaemic penumbra following middle cerebral artery occlusion (MCAO) in the Sprague Dawley rat. R18 (300 or 1000 nmol/kg), R18D (300 nmol/kg) or saline were administered intravenously 10 min after MCAO induced using a filament. Serial perfusion and diffusion-weighted MRI imaging was performed to measure changes in the infarct core and penumbra from time points between 45- and 225-min post-occlusion. Repeated measures analyses of infarct growth and penumbral tissue size were evaluated using generalised linear mixed models (GLMMs). R18D (300 nmol/kg) was most effective in slowing infarct core growth (46.8 mm3 reduction; p < 0.001) and preserving penumbral tissue (21.6% increase; p < 0.001), followed by R18 at the 300 nmol/kg dose (core: 29.5 mm3 reduction; p < 0.001, penumbra: 12.5% increase; p < 0.001). R18 at the 1000 nmol/kg dose had a significant impact in slowing core growth (19.5 mm3 reduction; p = 0.026), but only a modest impact on penumbral preservation (6.9% increase; p = 0.062). The in vitro anti-excitotoxic neuroprotective efficacy of R18D was also demonstrated to be unaffected when preincubated for 1-3 h or overnight, in a cell lysate prepared from dying neurons or with the proteolytic enzyme, plasmin, whereas the neuroprotective efficacy of R18 was significantly reduced after a 2-h incubation. These findings highlight the capacity of poly-arginine peptides to reduce infarct growth and preserve the ischaemic penumbra, and confirm the superior efficacy and proteolytic stability of R18D, which indicates that this peptide is likely to retain its neuroprotective properties when co-administered with alteplase during thrombolysis for acute ischaemic stroke.

Poly-arginine peptides reduce infarct volume in a permanent middle cerebral artery rat stroke model.

BACKGROUND: We recently reported that poly-arginine peptides have neuroprotective properties both in vitro and in vivo. In cultured cortical neurons exposed to glutamic acid excitotoxicity, we demonstrated that neuroprotective potency increases with polymer length plateauing at R15 to R18 (R = arginine resides). In an in vivo study in rats, we also demonstrated that R9D (R9 peptide synthesised with D-isoform amino acids) administered intravenously at a dose of 1000 nmol/kg 30 min after permanent middle cerebral artery occlusion (MCAO) reduces infarct volume. Based on these positive in vitro and in vivo findings, we decided to examine the neuroprotective efficacy of the L-isoform poly-arginine peptides, R12, R15 and R18 when administered at a dose of 1000 nmol/kg 30 min after permanent MCAO in the rat. RESULTS: At 24 h post-MCAO, there was reduced total infarct volume for R12 (12.8 % reduction) and R18 (20.5 % reduction), but this reduction only reached statistical significance for R18. Brain slice analysis revealed significantly reduced injury in coronal slices 4 and 5 for R18, and slice 5 for R12. The R15 peptide had no effect on infarct volume. Peptide treatment did not reveal any statistical significant improvement in functional outcomes. CONCLUSION: While these findings confirm the in vivo neuroprotective properties of poly-arginine peptides, additional dose studies are required particularly in less severe transient MCAO models so as to further assess the potential of these agents as a stroke therapy.

Case of fake acute abdomen by metastatic melanoma.

Malignant melanoma is the neoplasm with highest probability of cardiac metastatization. Cardiac involvement by metastatic melanoma is rarely identified ante-mortem (5-30% of cases) for non-specificity of cardiac symptoms. In fact we show in this case report that abdominal pain can represent the predominant symptom. Furthermore we show the importance of linkage between clinical & anamnestic data which if underestimated can lead to an improper management and to the patient exitus.

Ghost Ileostomy with or without abdominal parietal split.

BACKGROUND: In patients who undergo low anterior rectal resection, the fashioning of a covering stoma (CS) is still controversial. In fact, a covering stoma (ileostomy or colostomy) is worsened by major complications related to the procedure, longer recovery time, necessity of a re-intervention under general anesthesia for stoma closure and poorer quality of life. The advantage of Ghost Ileostomy (GI) is that an ileostomy can be performed only when there is clinical evidence of anastomotic leakage, without performing further interventions with related complications when anastomotic leak is absent and therefore the procedure is not necessary. Moreover, in case of anastomotic dehiscence and necessity of delayed stoma opening, mortality and morbidity in patients with GI are comparable with the ones that occur in patients which had a classic covering stoma. On the other hand, is simple to think about the possible economic saving: avoiding an admission for performing the closure of the ileostomy, with all the costs connected (OR, hospitalization, post-operative period, treatment of possible complications) represents a huge saving for the hospital management and also raise the quality of life of the patients. METHODS: In this study we prospectively analyzed 20 patients who underwent anterior extra-peritoneal rectum resection for rectal carcinoma with TME and fashioning of GI realized with or without abdominal parietal split. RESULTS: In the group of patients that received a GI without split laparotomy mortality was absent and in one case an anastomotic leak occurred. In the group of patients in which GI with split laparotomy was fashioned, one death occurred and there were one case of infection and one respiratory complication. Clinical follow-up was 12 months. CONCLUSIONS: The use of different techniques for fashioning a GI do not present significant differences when they are performed by expert surgeons, but further evidence is needed with more randomized trials, in order to have more data supporting the clinical observation.

Comparison of neuroprotective efficacy of poly-arginine R18 and R18D (D-enantiomer) peptides following permanent middle cerebral artery occlusion in the Wistar rat and in vitro toxicity studies.

We have previously demonstrated that arginine-rich and poly-arginine peptides possess potent neuroprotective properties, with poly-arginine peptide R18 identified as being highly effective at reducing infarct volume following middle cerebral artery occlusion (MCAO) in the Sprague Dawley rat. Since peptides synthesised using D-isoform amino acids have greater stability than L-isoform peptides due to increased resistance to proteolytic degradation, they represent potentially more effective peptide therapeutics. Therefore we compared the neuroprotective efficacy of R18 and its D-enantiomer R18D following permanent MCAO in the Wistar rat. Furthermore, as increased peptide stability may also increase peptide toxicity, we examined the effects of R18 and R18D on cultured cortical neurons, astrocytes, brain endothelial cells (bEND.3), and embryonic kidney cells (HEK293) following a 10-minute or 24-hour peptide exposure duration. The in vivo studies demonstrated that R18D resulted in a greater reduction in mean infarct volume compared to R18 (33%, p = 0.004 vs 12%, p = 0.27) after intravenous administration at 300 nmol/kg 30 minutes after MCAO. Both R18D and R18 reduced cerebral hemisphere swelling to a comparable degree (27%, p = 0.03 and 30%, p = 0.02), and improved neurological assessment scores (1.5, p = 0.02 and 2, p = 0.058 vs 3 for vehicle). No abnormal histological findings specific to peptide treatments were observed in hematoxylin and eosin stained sections of kidney, liver, spleen, lung and heart. In vitro studies demonstrated that R18 and R18D were most toxic to neurons, followed by astrocytes, HEK293 and bEND.3 cells, but only at high concentrations and/or following 24-hour exposure. These findings further highlight the neuroprotective properties of poly-arginine peptides, and indicate that R18D at the dose examined is more potent than R18 in Wistar rats, and justify continued investigation of the R18 peptide as a novel neuroprotective agent for stroke.

Delayed 2-h post-stroke administration of R18 and NA-1 (TAT-NR2B9c) peptides after permanent and/or transient middle cerebral artery occlusion in the rat.

Following positive results with the poly-arginine peptide R18 when administered intravenously 30 or 60min after permanent and/or transient middle cerebral artery occlusion (MCAO; 90min) in the rat, we examined the effectiveness of the peptide when administered 2h after MCAO. R18 was administered intravenously (1000nmol/kg via jugular vein) after permanent MCAO or a transient 3-h MCAO or when administered intra-arterially (100nmol/kg via internal carotid artery) immediately after reperfusion following a transient 2-h MCAO. In the transient MCAO studies, the neuroprotective NA-1 peptide was used as a positive control. Infarct volume, cerebral edema and functional outcomes were measured 24h after MCAO. Following permanent or transient MCAO, neither R18 nor NA-1 significantly reduced infarct volume. However, following permanent MCAO, R18 appeared to reduce cerebral edema (p=0.006), whereas following a transient 3-h MCAO, R18 improved the time to remove adhesive tape (p=0.04) without significantly affecting cerebral edema. There was also a trend (p=0.07) towards improved rota-rod performance with R18 in both permanent and transient 3-h MCAO. Following a transient 2-h MCAO, R18 had no significant effects on cerebral edema or neurological score but did lessen the extent of weight loss. Overall, while R18 had no effect on infarct volume, the peptide reduced cerebral edema after permanent MCAO, and improved some functional outcomes after transient MCAO.

Neuroprotective efficacy of poly-arginine R18 and NA-1 (TAT-NR2B9c) peptides following transient middle cerebral artery occlusion in the rat.

We examined the efficacy of R18 in a transient MCAO model and compared its effectiveness to the well-characterized neuroprotective NA-1 peptide. R18 and NA-1 peptides were administered intravenously (30, 100, 300, 1000nmol/kg), 60min after the onset of 90min of MCAO. Infarct volume, cerebral swelling and functional outcomes (neurological score, adhesive tape and rota-rod) were measured 24h after MCAO. R18 reduced total infarct volume by 35.1% (p=0.008), 24.8% (p=0.059), 12.2% and 9.6% for the respective 1000 to 30nmol/kg doses, while the corresponding doses of NA-1 reduced lesion volume by 26.1% (p=0.047), 16.6%, 16.5% and 7%, respectively. R18 also reduced hemisphere swelling by between 46.1% (1000 and 300nmol/kg; p=0.009) and 24.4% (100nmol/kg; p=0.066), while NA-1 reduced swelling by 25.7% (1000nmol/kg; p=0.054). In addition, several R18 and NA-1 treatment groups displayed a significant improvement in at least one parameter of the adhesive tape test. These results confirm the neuroprotective properties of R18, and suggest that the peptide is a more effective neuroprotective agent than NA-1. This provides strong justification for the continuing development of R18 as a neuroprotective treatment for stroke.

Assessment of the Neuroprotective Effects of Arginine-Rich Protamine Peptides, Poly-Arginine Peptides (R12-Cyclic, R22) and Arginine-Tryptophan-Containing Peptides Following In Vitro Excitotoxicity and/or Permanent Middle Cerebral Artery Occlusion in Rats.

We have demonstrated that arginine-rich and poly-arginine peptides possess potent neuroprotective properties with arginine content and peptide positive charge being particularly critical for neuroprotective efficacy. In addition, the presence of other amino acids within arginine-rich peptides, as well as chemical modifications, peptide length and cell-penetrating properties also influence the level of neuroprotection. Against this background, we have examined the neuroprotective efficacy of arginine-rich protamine peptides, a cyclic (R12-c) poly-arginine peptide and a R22 poly-arginine peptide, as well as arginine peptides containing tryptophan or other amino acids (phenylalanine, tyrosine, glycine or leucine) in in vitro glutamic acid excitotoxicity and in vivo rat permanent middle cerebral artery occlusion models of stroke. In vitro studies demonstrated that protamine and poly-arginine peptides (R12-c, R22) were neuroprotective. Arginine-tryptophan-containing peptides were highly neuroprotective, with R12W8a being the most potent arginine-rich peptide identified in our laboratory. Peptides containing phenylalanine or tyrosine substituted in place of tryptophan in R12W8a were also highly neuroprotective, whereas leucine, and in particular glycine substitutions, decreased peptide efficacy. In vivo studies with protamine administered intravenously at 1000 nmol/kg 30 min after MCAO significantly reduced infarct volume and cerebral oedema by 22.5 and 38.6%, respectively. The R12W8a peptide was highly toxic when administered intravenously at 300 or 100 nmol/kg and ineffective at reducing infarct volume when administered at 30 nmol/kg 30 min after MCAO, unlike R18 (30 nmol/kg), which significantly reduced infarct volume by 20.4%. However, both R12W8a and R18 significantly reduced cerebral oedema by 19.8 and 42.2%, respectively. Protamine, R12W8a and R18 also reduced neuronal glutamic acid-induced calcium influx. These findings further highlight the neuroprotective properties of arginine-rich peptides and support the view that they represent a new class of neuroprotective agent.

Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties.

Several recent studies have demonstrated that TAT and other arginine-rich cell penetrating peptides (CPPs) have intrinsic neuroprotective properties in their own right. Examples, we have demonstrated that in addition to TAT, poly-arginine peptides (R8 to R18; containing 8-18 arginine residues) as well as some other arginine-rich peptides are neuroprotective in vitro (in neurons exposed to glutamic acid excitotoxicity and oxygen glucose deprivation) and in the case of R9 in vivo (after permanent middle cerebral artery occlusion in the rat). Based on several lines of evidence, we propose that this neuroprotection is related to the peptide's endocytosis-inducing properties, with peptide charge and arginine residues being critical factors. Specifically, we propose that during peptide endocytosis neuronal cell surface structures such as ion channels and transporters are internalised, thereby reducing calcium influx associated with excitotoxicity and other receptor-mediated neurodamaging signalling pathways. We also hypothesise that a peptide cargo can act synergistically with TAT and other arginine-rich CPPs due to potentiation of the CPPs endocytic traits rather than by the cargo-peptide acting directly on its supposedly intended intracellular target. In this review, we systematically consider a number of studies that have used CPPs to deliver neuroprotective peptides to the central nervous system (CNS) following stroke and other neurological disorders. Consequently, we critically review evidence that supports our hypothesis that neuroprotection is mediated by carrier peptide endocytosis. In conclusion, we believe that there are strong grounds to regard arginine-rich peptides as a new class of neuroprotective molecules for the treatment of a range of neurological disorders.

Laparoscopic peritoneal lavage: a definitive treatment for diverticular peritonitis or a "bridge" to elective laparoscopic sigmoidectomy?: a systematic review.

To this day, the treatment of generalized peritonitis secondary to diverticular perforation is still controversial. Recently, in patients with acute sigmoid diverticulitis, laparoscopic lavage and drainage has gained a wide interest as an alternative to resection. Based on this backdrop, we decided to perform a systematic review of the literature to evaluate the safety, feasibility, and efficacy of peritoneal lavage in perforated diverticular disease.A bibliographic search was performed in PubMed for case series and comparative studies published between January 1992 and February 2014 describing laparoscopic peritoneal lavage in patients with perforated diverticulitis.A total of 19 articles consisting of 10 cohort studies, 8 case series, and 1 controlled clinical trial met the inclusion criteria and were reviewed. In total these studies analyzed data from 871 patients. The mean follow-up time ranged from 1.5 to 96 months when reported. In 11 studies, the success rate of laparoscopic peritoneal lavage, defined as patients alive without surgical treatment for a recurrent episode of diverticulitis, was 24.3%. In patients with Hinchey stage III diverticulitis, the incidence of laparotomy conversion was 1%, whereas in patients with stage IV it was 45%. The 30-day postoperative mortality rate was 2.9%. The 30-day postoperative reintervention rate was 4.9%, whereas 2% of patients required a percutaneous drainage. Readmission rate after the first hospitalization for recurrent diverticulitis was 6%. Most patients who were readmitted (69%) required redo surgery. A 2-stage laparoscopic intervention was performed in 18.3% of patients.Laparoscopic peritoneal lavage should be considered an effective and safe option for the treatment of patients with sigmoid diverticulitis with Hinchey stage III peritonitis; it can also be consider as a "bridge" surgical step combined with a delayed and elective laparoscopic sigmoidectomy in order to avoid a Hartmann procedure. This minimally invasive staged approach should be considered for patients without systemic toxicity and in centers experienced in minimally invasive surgery techniques. Further evidence is needed, and the ongoing RCTs will better define the role of the laparoscopic peritoneal lavage/drainage in the treatment of patients with complicated diverticulitis.

The sigmoid volvulus: surgical timing and mortality for different clinical types.

BACKGROUND: In western countries intestinal obstruction caused by sigmoid volvulus is rare and its mortality remains significant in patients with late diagnosis. The aim of this work is to assess what is the correct surgical timing and how the prognosis changes for the different clinical types. METHODS: We realized a retrospective clinical study including all the patients treated for sigmoid volvulus in the Department of General Surgery, St Maria Hospital, Terni, from January 1996 till January 2009. We selected 23 patients and divided them in 2 groups on the basis of the clinical onset: patients with clear clinical signs of obstruction and patients with subocclusive symptoms. We focused on 30-day postoperative mortality in relation to the surgical timing and procedure performed for each group. RESULTS: In the obstruction group mortality rate was 44% and it concerned only the patients who had clinical signs and symptoms of peritonitis and that were treated with a sigmoid resection (57%). Conversely none of the patients treated with intestinal derotation and colopexy died. In the subocclusive group mortality was 35% and it increased up to 50% in those patients with a late diagnosis who underwent a sigmoid resection. CONCLUSIONS: The mortality of patients affected by sigmoid volvulus is related to the disease stage, prompt surgical timing, functional status of the patient and his collaboration with the clinicians in the pre-operative decision making process. Mortality is higher in both obstructed patients with generalized peritonitis and patients affected by subocclusion with late diagnosis and surgical treatment; in both scenarios a Hartmann's procedure is the proper operation to be considered.

Fibrin glue in the treatment of anal fistula: a systematic review.

BACKGROUND: New sphincter-saving approaches have been applied in the treatment of perianal fistula in order to avoid the risk of fecal incontinence. Among them, the fibrin glue technique is popular because of its simplicity and repeatability. The aim of this review is to compare the fibrin glue application to surgery alone, considering the healing and complication rates. METHODS: We performed a systematic review searching for published randomized and controlled clinical trials without any language restriction by using electronic databases. All these studies were assessed as to whether they compared conventional surgical treatment versus fibrin glue treatment in patients with anal fistulas, in order to establish both the efficacy and safety of each treatment. We used Review Manager 5 to conduct the review. RESULTS: The healing rate is higher in those patients who underwent the conventional surgical treatment (P = 0,68), although the treatment with fibrin glue gives no evidence of anal incontinence (P = 0,08). Furthermore two subgroup analyses were performed: fibrin glue in combination with intra-adhesive antibiotics versus fibrin glue alone and anal fistula plug versus fibrin glue. In the first subgroup there were not differences in healing (P = 0,65). Whereas in the second subgroup analysis the healing rate is statistically significant for the patients who underwent the anal fistula plug treatment instead of the fibrin glue treatment (P = 0,02). CONCLUSION: In literature there are only two randomized controlled trials comparing the conventional surgical management versus the fibrin glue treatment in patients with anal fistulas. Although from our statistical analysis we cannot find any statistically significant result, the healing rate remains higher in patients who underwent the conventional surgical treatment (P = 0,68), and the anal incontinence rate is very low in the fibrin glue treatment group (P = 0,08). Anyway the limited collected data do not support the use of fibrin glue. Moreover, in our subgroup analysis the use of fibrin glue in combination with intra-adhesive antibiotics does not improve the healing rate (P = 0.65), whereas the anal fistula plug treatment compared to the fibrin glue treatment shows good results (P = 0,02), although the poor number of patients treated does not lead to any statistically evident conclusion. This systematic review underlines the need of new RCTs upon this issue.

Virtual colonoscopy in stenosing colorectal cancer.

BACKGROUND: Between 5 and 10% of the patients undergoing a colonoscopy cannot have a complete procedure mainly due to stenosing neoplastic lesion of rectum or distal colon. Nevertheless the elective surgical treatment concerning the stenosis is to be performed after the pre-operative assessment of the colonic segments upstream the cancer. The aim of this study is to illustrate our experience with the Computed Tomographic Colonography (CTC) for the pre-operative assessment of the entire colon in the patients with stenosing colorectal cancers. METHODS: From January 2005 till March 2009, we observed and treated surgically 43 patients with stenosing colorectal neoplastic lesions. All patients did not tolerate the pre-operative colonoscopy. For this reason they underwent a pre-operative CTC in order to have a complete assessment of the entire colon. All patients underwent a follow-up colonoscopy 3 months after the surgical treatment. The CTC results were compared with both macroscopic examination of the specimen and the follow-up coloscopy. RESULTS: The pre-operative CTC showed four synchronous lesions in four patients (9.3% of the cases). The macroscopic examination of the specimen revealed three small sessile polyps (3-4 mm in diameter) missed in the pre-operative assessment near the stenosing colorectal cancer. The follow-up colonoscopy showed four additional sessile polyps with a diameter between 3-11 mm in three patients. Our experience shows that CTC has a sensitivity of 83,7%. CONCLUSION: In patients with stenosing colonic lesions, CTC allows to assess the entire colon pre-operatively avoiding the need of an intraoperative colonoscopy. More synchronous lesions are detected and treated at the time of the elective surgery for the stenosing cancer avoiding further surgery later on.

Emergency treatment of complicated incisional hernias: a case study.

BACKGROUND: The emergency treatment of incisional hernias is infrequent but it can be complicated with strangulation or obstruction and in some cases the surgical approach may also include an intestinal resection with the possibility of peritoneal contamination. Our study aims at reporting our experience in the emergency treatment of complicated incisional hernias. METHODS: Since January 1999 till July 2008, 89 patients (55 males and 34 females) were treated for complicated incisional hernias in emergency. The patients were divided in two groups: Group I consisting of 33 patients that were treated with prosthesis apposition and Group II, consisting of 56 patients that were treated by performing a direct abdominal wall muscles suture. RESULTS: All the patients underwent a 6-month follow up; we noticed 9 recurrences (9/56, 16%) in the patients treated with direct abdominal wall muscles suture and 1 recurrence (1/33, 3%) in the group of patients treated with the prosthesis apposition. CONCLUSIONS: According to our experience, the emergency treatment of complicated incisional hernias through prosthesis apposition is always feasible and ensures less post-operative complications (16% vs 21,2%) and recurrences (3% vs 16%) compared to the patients treated with direct muscular suture.

A severe case of hemobilia and biliary fistula following an open urgent cholecystectomy.

BACKGROUND: Cholecystectomy has been the treatment of choice for symptomatic gallstones, but remains the greatest source of post-operative biliary injuries. Laparoscopic approach has been recently preferred because of short hospitalisation and low morbidity but has an higher incidence of biliary leakages and bile duct injuries than open one due to a technical error or misinterpretation of the anatomy. Even open cholecystectomy presents a small number of complications especially if it was performed in urgency. Hemobilia is one of the most common cause of upper gastrointestinal bleeding from the biliary ducts into the gastrointestinal tract due to trauma, advent of invasive procedures such as percutaneous liver biopsy, transhepatic cholangiography, and biliary drainage. METHODS: We report here a case of massive hemobilia in a 60-year-old man who underwent an urgent open cholecystectomy and a subsequent placement of a transhepatic biliary drainage. CONCLUSION: The management of these complications enclose endoscopic, percutaneous and surgical therapies. After a diagnosis of biliary fistula, it's most important to assess the adequacy of bile drainage to determine a controlled fistula and to avoid bile collection and peritonitis. Transarterial embolization is the first line of intervention to stop hemobilia while surgical intervention should be considered if embolization fails or is contraindicated.