Assessing the risk of performance and detection bias in Cochrane reviews as a joint domain is less accurate compared to two separate domains.

BACKGROUND: Initially, the Cochrane risk of bias (RoB) tool had a domain for "blinding of participants, personnel and outcome assessors". In the 2011 tool, the assessment of blinding was split into two domains: blinding of participants and personnel (performance bias) and blinding of outcome assessors (detection bias). The aims of this study were twofold; first, to analyze the frequency of usage of the joint blinding domain (a single domain for performance and detection bias), and second, to assess the proportion of adequate assessments made in the joint versus single RoB domains for blinding by comparing whether authors' RoB judgments were supported by explanatory comments in line with the Cochrane Handbook recommendations. METHODS: We extracted information about the assessment of blinding from RoB tables (judgment, comment, and whether it was specified which outcome type; e.g., objective, subjective) of 729 Cochrane reviews published in 2015-2016. In the Cochrane RoB tool, judgment (low, unclear or high risk) needs to be accompanied by a transparent comment, in which authors provide a summary justifying RoB judgment, to ensure transparency in how these judgments were reached. We reassessed RoB based on the supporting comments reported in Cochrane RoB tables, in line with instructions from the Cochrane Handbook. Then, we compared our new assessments to judgments made by Cochrane authors. We compared the frequency of adequate judgments in reviews with two separate domains for blinding versus those with a joint domain for blinding. RESULTS: The total number of assessments for performance bias was 6918, with 8656 for detection bias and 3169 for the joint domain. The frequency of adequate assessments was 74% for performance bias, 78% for detection bias, and 59% for the joint domain. The lowest frequency of adequate assessments was found when Cochrane authors judged low risk - 47% in performance bias, 62% in detection bias, and 31% in the joint domain. The joint domain and detection bias domain had a similar proportion of specified outcome types (17% and 18%, respectively). CONCLUSIONS: Splitting joint RoB assessment about blinding into two domains was justified because the frequency of adequate judgments was higher in separate domains. Specification of outcome types in RoB domains should be further scrutinized.

Predictive value of abdominal drain amylase for postoperative pancreatic fistula following gastric resection: A systematic review and meta-analysis.

BACKGROUND: Radical dissection of lymph nodes, accompanying gastric cancer resection, can lead to collateral damage to the pancreas and development of postoperative pancreatic fistula (POPF). METHODS: We searched the Cochrane Library, MEDLINE, Embase, and Web of Science up to April 21, 2020, to identify studies documenting the value of abdominal drain amylase level (d-AMY) on postoperative day 1 (POD1) as a predictor of POPF after gastric surgery. The quality of selected studies was assessed using the QUADAS-2 tool. The diagnostic value of d-AMY on POD1 for prediction of POPF was first assessed by calculation of pooled estimates of sensitivity, specificity, likelihood ratios (LR), and the diagnostic odds ratio (DOR). Secondly, the accuracy was further demonstrated graphically with the hierarchical summary receiver operating curve (hSROC). PROSPERO registration number: CRD42020181145. RESULTS: DOR of nine studies (cases n = 1856) observing the occurrence of POPF after measurement of d-AMY on POD1 was 18.7 (95%CI: 10.0, 34.8), and the area under hSROC was 0.88 ± 0.02. The pooled sensitivity was 0.74 (95%CI: 0.66, 0.81) and specificity 0.84 (95%CI: 0.82, 0.86). The negative LR was at the lowest point of 0.16 (95%CI: 0.07, 0.37) at the cutoff value for d-AMY of 941 IU/L, while the positive LR ranged from 4.4 (cutoff 2119 IU/L) to 6.2 (cutoff 5000 IU/L). CONCLUSION: d-AMY on POD1 can be used as an accurate and non-invasive predictor of POPF in the earliest stage of postoperative course following gastric cancer resection; value ≤ 941 IU/L warrants early drain removal and low probability of POPF (any grade).

Adequacy of risk of bias assessment in surgical vs non-surgical trials in Cochrane reviews: a methodological study.

BACKGROUND: Bias in randomized controlled trials (RCTs) can lead to underestimation or overestimation of the true effects of interventions. Surgical RCTs may suffer from the risk of bias (RoB) that is avoidable in trials of other interventions, and vice versa. We aimed to compare the adequacy of RoB assessments in surgical versus non-surgical RCTs included in Cochrane reviews and to assess the most common differences in those RoB assessments. Due to specificities of surgical trials, i.e. difficulties associated with blinding of surgical interventions, we hypothesized that assessments of surgical trials may be more adequate, compared to RCTs of non-surgical interventions. METHODS: This was a methodological study, analyzing methods of published Cochrane systematic reviews. Data were extracted from RoB tables in Cochrane reviews (judgments and accompanying explanatory comment) for the following four RoB domains used in the 2011 Cochrane RoB tool: randomization, allocation concealment, blinding of participants and personnel, and blinding of outcome assessors. We defined adequate assessments as those that were in line with instructions from the Cochrane Handbook for Systematic Reviews of Interventions. The prevalence of adequate assessments was compared in surgical versus non-surgical trials. The most common differences in both groups of reviews were presented. RESULTS: In 729 analyzed Cochrane reviews, there were 10,537 included trials. The prevalence of adequate RoB judgments made by Cochrane authors ranged from 87.9, 95%CI (87.3 to 88.6%) for randomization to 70.7, 95%CI (69.8 to 71.5%) for blinding of participants and personnel. For all analyzed RoB domains, the prevalence of adequate RoB domains was higher in surgical trials than in non-surgical trials. For two RoB domains assessing blinding, this difference between surgical and non-surgical trials was statistically significant (P < 0.001), while the difference was not significant for the RoB domain regarding randomization (P = 0.124) and allocation concealment (P = 0.039, ß < 0.8). CONCLUSIONS: RoB judgments were more in line with instructions from the Cochrane Handbook when Cochrane reviews assessed surgical trials, compared to those that analyzed non-surgical interventions. However, further steps are warranted to scrutinize RoB assessment in trials of both surgical and non-surgical interventions.

Discordant outcomes of laparoscopic versus open appendectomy for suspected appendicitis during pregnancy in published meta-analyses: an overview of systematic reviews.

OBJECTIVES: Our study aimed to explore the discrepancy of results between overlapping systematic reviews (SRs) of laparoscopic appendectomy (LA) versus open appendectomy (OA) for suspected appendicitis during pregnancy. METHODS: MEDLINE, Embase, and Cochrane Database of Systematic Reviews (CDSR) were searched for SRs published from January 1, 2017 to September 10, 2019. SRs and meta-analyses (MA) that compared outcomes of LA versus OA during pregnancy were used. Data regarding the methodology of SR/MA, included studies, efficacy and safety outcomes were extracted. SR quality was analysed with AMSTAR 2. RESULTS: Four SRs were found, published between April 2018 and April 2019. These reviews included a variety of primary studies, ranging from 17 to 22, and number of included patients varied from 4694 to 6276. A total of 13 outcomes were analyzed. Nine outcomes were included in more than one review; among them, discrepancies between summary effect sizes in meta-analyses were found in four outcomes: preterm birth, Apgar score at 5 min, length of stay in hospital, and wound infection rates. One primary study, which included more than half of the total number of patients in analyzed reviews, showed a predominant effect on the outcome for fetal loss. All four SRs had critically low methodological quality. CONCLUSIONS: The outcomes of LA versus OA for suspected appendicitis in pregnancy represented in four recent SRs do not provide consistent results. Such uncertainties require new, high-quality primary and secondary evidence on this topic.

Assessing risk of bias judgments for blinding of outcome assessors in Cochrane reviews.

Aim: Adequate judging of risk of bias (RoB) for blinding of outcome assessors (detection bias) is important for supporting highest level of evidence. Materials & methods: Judgments and supporting comments for detection bias were retrieved from RoB tables reported in Cochrane reviews. We categorized comments, and then compared judgment and supporting comment with instructions from the Cochrane Handbook. Results: We analyzed 8656 judgments for detection bias from 7626 trials included in 575 reviews. Overall, 1909 judgments (22%) were not in line with the Cochrane Handbook. In 9% of trials, the authors split the detection bias domain according to outcomes. Here, prevalence of inadequate judgments was 19%. Conclusion: Interventions to improve RoB assessments in systematic reviews should be explored.

Risk of bias judgments for random sequence generation in Cochrane systematic reviews were frequently not in line with Cochrane Handbook.

BACKGROUND: Assessing the risk of bias (RoB) in included studies is one of the key methodological aspects of systematic reviews. Cochrane systematic reviews appraise RoB of randomised controlled trials (RCTs) with the Cochrane RoB tool. Detailed instructions for using the Cochrane RoB tool are provided in the Cochrane Handbook for Systematic Reviews of Interventions (The Cochrane Handbook). The purpose of this study was to analyse whether Cochrane authors use adequate judgments about the RoB for random sequence generation of RCTs included in Cochrane reviews. METHODS: We extracted authors' judgments (high, low or unclear RoB) and supports for judgments (comments accompanying judgments which explain the rationale for a judgment) for random sequence generation of included RCTs from RoB tables of Cochrane reviews using automated data scraping. We categorised all supporting comments, analysed the number and type of various supporting comments and assessed adequacy of RoB judgment for randomisation in line with recommendations from the Cochrane Handbook. RESULTS: We analysed 10,103 RCTs that were included in 704 Cochrane reviews. For 5,706 RCTs, randomisation was not described, but for the remaining RCTs, it was indicated that randomisation was performed using computer/software/internet (N = 2,850), random number table (N = 883), mechanical method (N = 359) or it was incomplete/inappropriate (N = 305). Overall, 1,220/10,103 trials (12%) did not have a RoB judgment in line with Cochrane Handbook guidance about randomisation. The highest proportion of misjudgements was found for trials with high RoB (28%), followed by those with low (20%) or unclear (3%). Therefore, one in eight judgments for the analysed domain in Cochrane reviews was not in line with Cochrane Handbook, and one in four if the judgment was "high risk". CONCLUSION: Authors of Cochrane reviews often make judgments about the RoB related to random sequence generation that are not in line with instructions given in the Cochrane Handbook, which compromises the reliability of the systematic reviews. Our results can help authors of both Cochrane and non-Cochrane reviews which use Cochrane RoB tool to avoid making common mistakes when assessing RoB in included trials.

Risk of bias assessments for blinding of participants and personnel in Cochrane reviews were frequently inadequate.

OBJECTIVES: The objective of this study was to analyze adequacy of judgments about risk of bias (RoB) for blinding of participants and personnel (performance bias) in Cochrane systematic reviews of randomized controlled trials (RCTs). STUDY DESIGN AND SETTING: We extracted judgments and supporting comments for performance bias from Cochrane reviews' RoB tables using automated data scraping. We parsed all intervention descriptions, judgments about risk of performance bias, and comments supporting judgments into simple categories. We assessed adequacy of RoB judgments against recommendations from the Cochrane Handbook. RESULTS: We analyzed judgments for performance bias of 10,429 RCTs included in 718 Cochrane reviews. Overall, 1,828 out of 6,918 judgments (26%) for performance bias were not in line with the Cochrane Handbook and were therefore considered inadequate. In reviews where Cochrane authors have split the performance bias domain into two subdomains, based on blinded individuals, we found lower prevalence of inadequate risk of bias judgments, with 9% of judgments for blinding of participants, and 5.8% judgments for the blinding of personnel subdomain being judged inadequately. CONCLUSION: In Cochrane reviews, risk of bias assessments for blinding of participants and personnel were frequently not in line with Cochrane Handbook recommendations. Interventions to improve these assessments should be taken into consideration.

Outcome domains and pain outcome measures in randomized controlled trials of interventions for postoperative pain in children and adolescents.

BACKGROUND: We analysed outcome domains and pain outcome measures in randomized controlled trials of interventions for postoperative pain management in children and adolescents and compared them to the core outcome set recommended by the Pediatric Initiative on Methods, Measurement and Pain Assessment in Clinical Trials (PedIMMPACT). METHODS: Systematic literature search was conducted in MEDLINE, CDSR, DARE, CINAHL and PsycINFO up to 31 January 2017. One author extracted data and second verified the extraction. Outcome domains and pain outcome measures were analysed and compared with the PedIMMPACT core outcome set. RESULTS: We included 337 trials. Median number of reported outcomes was five (range 1-11) for the included trials and two (range 0-6) for PedIMMPACT. The most commonly analysed PedIMMPACT outcome domains were pain intensity (93%) and "symptoms and adverse events" (83%). The remaining four PedIMMPACT outcomes were present in under 30% of included randomized controlled trials. Proportion of PedIMMPACT outcome domains did not change after the PedIMMPACT was published in 2008. Of the 312 trials that reported pain intensity, 303 (97%) also specified pain assessment tools, in which the most common was the visual analogue scale (24%) followed by the Children's Hospital of Eastern Ontario Pain Scale (18%). CONCLUSION: Analysed trials about interventions for pediatric postoperative pain insufficiently used the recommended core outcome set for acute pain in children. Relevance of the PedIMMPACT core outcome set, as well as the reasons behind its limited uptake, need to be further evaluated. SIGNIFICANCE: Recommended core outcomes have been insufficiently used in randomized controlled trials about postoperative pain in children, which hinders comparability of studies and makes synthesis of evidence difficult.

Authors' lack of awareness and use of core outcome set on postoperative pain in children is hindering comparative effectiveness research.

AIM: To analyze awareness about and acceptability of core outcome set (COS) for pediatric pain recommended by the PedIMMPACT. METHODS: We invited authors of systematic reviews and randomized controlled trials about interventions for postoperative pain in children to participate in a survey. RESULTS: Only a third of surveyed authors of systematic reviews and randomized controlled trials about postoperative pain in children had heard about the PedIMMPACT COS for acute pediatric pain. Problems indicated as preventing them from using the COS were lack of awareness, difficulties with implementation, and lack of resources. CONCLUSION: Further discussions about the adequacy of COS for acute pediatric pain, as well as interventions to increase the uptake of COS may be warranted.

Efficacy and Safety Outcomes in Systematic Reviews of Interventions for Postoperative Pain in Children: Comparison Against the Recommended Core Outcome Set.

Objective: To investigate the range of efficacy and safety outcomes used in systematic reviews (SRs) of randomized controlled trials (RCTs) of interventions for postoperative pain in children and compare them with outcome domains recommended in the Pediatric Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (PedIMMPACT). Methods: Five electronic databases were searched: MEDLINE, Cochrane Database of Systematic Reviews, DARE, CINAHL, and PsycINFO. Two review authors extracted outcome data independently. Efficacy and safety outcomes were extracted and categorized. The type and number of outcomes were analyzed and compared against the outcomes recommended by PedIMMPACT. The study protocol was registered in PROSPERO (CRD42015029654). Results: We included 48 systematic reviews with data from 816 trials. The median number of all outcomes was 4, while the median number of the PedIMMPACT core outcomes was three out of six. The most commonly reported outcome of the PedIMMPACT Core Outcome set (COS) was "symptoms and adverse events," followed by pain intensity, which was reported in 75% of the included SRs. Just over half of the SRs that included a pain intensity outcome also indicated the specific pain assessment tool used in the methods section. Conclusions: Systematic reviews in the field of pediatric pain do not use the recommended COS. Nor do they consistently include pain as an outcome. This makes comparisons of efficacy and safety across interventions very difficult. Future studies should explore whether the authors are aware of the COS and whether the recommended COS is appropriate.

Efficacy and Safety Outcome Domains and Outcome Measures in Systematic Reviews of Neuropathic Pain Conditions.

OBJECTIVES: Heterogeneity of outcome domains, used in interventional trials and systematic reviews (SRs) for neuropathic pain (NeuP), makes decisions on the comparative effectiveness of available treatments difficult. This study analyzed outcome domains and measures used in SRs of randomized controlled trials on efficacy and safety of interventions for NeuP and compared them with the core outcome set (COS) and core outcome measures (COMs) for chronic pain recommended by the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT). METHODS: Five electronic databases were searched to find SRs of interventions for NeuP. Outcome domains and measures were independently extracted by 2 authors, and compared against the IMMPACT-recommended COS and COMs. Outcome domains specified in the methods and reported in the results were also compared. RESULTS: Ninety-seven SRs were analyzed. The 2 core domains most frequently specified in the methods and reported in the results of SRs were pain and symptoms and adverse events. Pain intensity was mostly assessed with Visual Analog Scale (n=59) and Numerical Rating Scale (n=29). The incidence (n=70) and severity (n=60) were most commonly reported for adverse events. There were 240 different outcome measures used for the assessment of treatment efficacy and safety. CONCLUSIONS: Authors of SRs in the field of NeuP insufficiently use relevant recommended COS and COMs for chronic pain. More effort should be put into the implementation of COS to ensure that the study results can be compared and combined. There is a need for defining core outcome domains and measures specific for NeuP.

Anesthesia and perioperative pain management during cardiac electronic device implantation.

BACKGROUND: The degree of pain caused by the implantation of cardiac electronic devices (CEDs) and the type of anesthesia or perioperative pain management used with the procedure have been insufficiently studied. The aim of this study was to analyze perioperative pain management, as well as intensity and location of pain among patients undergoing implantation of CED, and to compare the practice with published guidelines. PATIENTS AND METHODS: This was a combined retrospective and prospective study conducted at the tertiary hospital, University Hospital Split, Croatia. The sample included 372 patients who underwent CED implantation. Perioperative pain management was analyzed retrospectively in 321 patients who underwent CED implantation during 2014. In a prospective study, intensity and location of pain before, during, and after the procedure were measured by using a numerical rating scale (NRS) ranging from 0 to 10 in 51 patients at the same institution from November 2014 to August 2015. RESULTS: A quarter of patients received analgesia or sedation before surgery. All the patients received local lidocaine anesthesia. After surgery, 31% of patients received pain medication or sedation. The highest pain intensity was observed during CED implantation with the highest NRS pain score being 8. Some patients reported severe pain (NRS >5) also at 1, 3, 6, 8, and 24 hours after surgery. The most common pain locations were surgical site, shoulder, and chest. Adherence to guidelines for acute perioperative pain management was insufficient. CONCLUSION: Patients may experience severe pain during and after CED implantation. Perioperative pain management was suboptimal, and higher doses of sedation and intensive analgesia are required. Guidelines for acute perioperative pain management and anesthesia during CED implantation should be developed.

Analysis of perioperative pain management in vascular surgery indicates that practice does not adhere with guidelines: a retrospective cross-sectional study.

BACKGROUND: Inadequate treatment of pain related to surgery may be associated with complications and prolonged recovery time and increased morbidity and mortality rates. We investigated perioperative pain management in vascular surgery and compared it with the relevant guidelines for the treatment of perioperative pain. METHODS: We conducted a retrospective study on 501 patients who underwent vascular surgery at the University Hospital Split, Croatia. We collected the following data from patients' charts: age, gender, premedication, preoperative patient's physical status, type of surgery, duration of surgery and anesthesia, type of anesthesia, postoperative analgesia, and need for intensive care. We examined departmental procedures to assess adherence to guidelines for perioperative pain management. RESULTS: None of the 501 patients' charts recorded information about perioperative pain intensity, 28% of patients did not receive any medication the night before their elective surgical procedures, and 17% of patients did not receive premedication immediately before the procedure. Most patients (66%) did not receive any pain medication in the operating room after surgery. Following surgery, 36% of patients were monitored in the intensive care units, while the rest were released to the ward. Some patients (17%) did not receive any analgesia after surgery. Procedures at the department did not adhere to the current recommendations for perioperative pain management. CONCLUSION: The study indicates that management of surgery-related pain in complex vascular procedures at this hospital did not follow guidelines for the management of acute perioperative pain. Our finding that most patients did not receive appropriate analgesia after vascular surgery leads to the conclusion that the institution would benefit from developing guidelines for the management of acute perioperative pain, which should be applied in all cases.

A Case of 2-Year-Old Child with Entero-Enteric Fistula Following Ingestion of 25 Magnets.

INTRODUCTION: Magnet ingestion usually does not cause serious complications, but in case of multiple magnet ingestion or ingestion of magnet with other metal it could cause intestinal obstruction, fistula formation or even perforation. CASE REPORT: We report case of intestinal obstruction and fistula formation following ingestion of 25 magnets in a 2-year-old girl. Intraoperatively omega shaped intestinal loop with fistula caused by two magnetic balls was found. Intestine trapped with magnetic balls was edematous and inflamed. Resection of intestinal segment was performed, followed by entero-enteric anastomosis. A total of 25 magnets were removed from resected intestine. CONCLUSION: Single magnet ingestion is treated as non-magnetic foreign body. Multiple magnet ingestion should be closely monitored and surgical approach could be the best option to prevent or to cure its complications.

Diabetes mellitus affects activity of calcium/calmodulin-dependent protein kinase II alpha in rat trigeminal ganglia.

The activity of calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory trigeminal neurons. The aim of this study was to investigate the immunoreactivity of phosphorylated CaMKIIα (pCaMKIIα) in subpopulations of trigeminal ganglion (TG) neurons in rat models of early diabetes type 1 (dm1) and 2 (dm2). DM1 model was induced with intraperitoneally (i.p.) injected streptozotocin (STZ) (55mg/kg). DM2 rats were fed with the high fat diet (HFD) for 2 weeks and then received 35mg/kg of STZ i.p. Two weeks and 2 months after the STZ-diabetes induction, rats were sacrificed and immunohistochemical analysis for detection of pCaMKIIα immunoreactivity and double immunofluorescence labelling with isolectin (IB4) was performed. Increased intensity of pCaMKIIα immunofluorescence, restricted to IB4-negative small-diameter neurons, was seen in TG neurons two months after STZ-DM1 induction. DM1 model, as well as the obesity (control dm2 groups) resulted in neuronal impaired growth while dm2 model led to neuron hypertrophy in TG. Observed changes may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory trigeminal neurons. In future, innovative strategies for modulation of CaMKIIα activity in specific subpopulations of neurons could be a novel approach in therapy of diabetic trigeminal neuropathy.

Cutaneous expression of calcium/calmodulin-dependent protein kinase II in rats with type 1 and type 2 diabetes.

Changes in calcium-calmodulin protein kinase II (CaMKII) have been well demonstrated in nervous tissue of diabetic animal models. Skin shares the same ectodermal origin as nervous tissue and it is often affected in diabetic patients. The goal of this study was to analyze expression of CaMKII in rat foot pad 2 weeks and 2 months after induction of diabetes type 1 and 2. Forty-two Sprague-Dawley rats were used. Diabetes mellitus type 1 (DM1) was induced with intraperitoneally (i.p.) injected 55 mg/kg of streptozotocin (STZ) and diabetes mellitus type 2 (DM2) with a combination of high-fat diet (HFD) and i.p. injection of low-dose STZ (35 mg/kg). Two weeks and two months following diabetes induction rats were sacrificed and skin samples from plantar surface of the both hind paws were removed. Immunohistochemistry was performed for detection of total CaMKII (tCaMKII) and its alpha isoform (pCaMKIIα). For detection of intraepidermal nerve fibers polyclonal antiserum against protein gene product 9.5 (PGP 9.5) was used. The results showed that CaMKII was expressed in the skin of both diabetic models. Total CaMKII was uniformly distributed throughout the epidermis and pCaMKIIα was limited to stratum granulosum. The tCaMKII and pCaMKIIα were not expressed in intraepidermal nerve fibers. Two weeks after induction of diabetes in rats there were no significant differences in expression of tCaMKII and pCaMKIIα between DM1 and DM2 compared to respective controls. In the 2-month experiments, significant increase in epidermal expression of tCaMKII and pCaMKIIα was observed in DM1 animals compared to controls, but not in DM2 animals. This study is the first description of cutaneous CaMKII expression pattern in a diabetic model. CaMKII could play a role in transformation of skin layers and contribute to cutaneous diabetic changes. Further research on physiological role of CaMKII in skin and its role in cutaneous diabetic complications should be undertaken in order to elucidate its function in epidermis.

The expression of calcium/calmodulin-dependent protein kinase II in the dorsal horns of rats with type 1 and type 2 diabetes.

The activation of calcium/calmodulin-dependent protein kinase II (CaMKII) has been proposed as a key factor in chronic pain development. This study therefore aimed to investigate the expression of CaMKII in the dorsal horn in a rat model of early phase diabetes mellitus (DM) types 1 and 2. Sprague-Dawley rats were used. DM1 was induced using streptozotocin (STZ) (55mg/kg injected intraperitoneally (i.p.)). DM2 was induced using a combination of a high fat diet (HFD) and STZ (35mg/kg i.p.). Controls received an i.p. injection of pure citrate buffer solution. DM2 animals and their controls also received HFD 2 weeks prior to the i.p. injection. Rats were sacrificed 2 weeks and 2 months after diabetes induction. The expression of tCaMKII, pCaMKIIα and IB4 in the dorsal horns was quantified using immunohistochemistry. Increased expression of tCaMKII and pCaMKIIα was seen in the dorsal horns of DM1 animals 2 weeks and 2 months after diabetes induction. In DM2 animals, similar changes in the expression of tCaMKII and pCaMKIIα were observed after 2 weeks, but not after 2 months. The expression of pCaMKIIα was most pronounced in laminae I-III. No difference in IB4 expression was observed between the groups. These results suggest a potential role for CaMKII in diabetic neuropathy development. Inhibition of CaMKII signaling pathways should be further explored as a potential treatment target in painful diabetic neuropathy.

Changes of calcium/calmodulin-dependent protein kinase II expression in dorsal root ganglia during maturation in long-term diabetes.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is considered one of the key intracellular signaling proteins for development of neuropathy. We analyzed the expression of total CaMKII (tCaMKII) and its alpha, beta, gamma and delta isoforms in dorsal root ganglia (DRG) in a rat model of Diabetes mellitus type I (DM1), 6 months and 1 year after diabetes induction. Diabetes was induced with streptozotocin and confirmed by measuring glucose levels and weight increase. Immunohistochemistry was performed for detection of tCaMKII and its isoforms in L4 and L5 DRGs. A significant decrease of CaMKII alpha and beta isoforms was noted 6 months after diabetes induction, while CaMKII gamma and delta were significantly decreased after 12 months in diabetic rats compared to controls. Analysis of neuronal subgroups based on the neuronal diameter revealed that the expression of alpha, beta and delta isoforms decreased only in small-diameter neurons. In conclusion, a significant decrease of specific CaMKII isoforms in small-diameter DRG neurons may suggest involvement of CaMKII alpha, beta and delta in the development of complex events responsible for the development of neuropathy in long-term diabetes during maturation. CaMKII is a part of the neuronal pathway that regulates the firing properties of excitable cells, especially neurons, and decreased CaMKII activity may be responsible for generation of aberrant signals, hyperalgesia and neuropathic pain.

Changes in epidermal thickness and cutaneous innervation during maturation in long-term diabetes.

AIM: Peripheral nerve fiber depletion in patients with chronic diabetes mellitus (DM) was linked to neuropathic symptoms, development of pain, foot ulcerations and lower extremity amputation. The aim of this study was to analyze cutaneous changes, including paw epidermal thickness and intraepidermal nerve fiber (IENF) density in long-term diabetes, in rats 6 months and 12 months after induction of diabetes. MATERIALS AND METHODS: Epidermal thickness and IENF density were studied in Sprague-Dawley diabetic rats 6 months and 12 months after diabetes induction with streptozotocin. Epidermal thickness was evaluated using hematoxylin and eosin staining. Peripheral nerve fibers were stained with polyclonal antiserum against protein gene product 9.5 (PGP 9.5). Successful diabetes induction was validated by measuring plasma glucose and body mass regularly throughout the experiment. RESULTS: This study showed that long-term diabetes, induced in Sprague-Dawley rats with streptozotocin, is characterized with significant epidermal thinning and reduction of intraepidermal nerve fibers, 6 months and 12 months after induction of diabetes. CONCLUSION: Long-term studies of streptozotocin models of diabetes could be used for making normative IENF densities that can be later used as age-dependent normative values for studying new treatment modalities.

Calcium/calmodulin-dependent protein kinase II in dorsal horn neurons in long-term diabetes.

The aim of this study was to investigate the expression of total calcium/calmodulin-dependent protein kinase II (CaMKII) and its phosphorylated α isoform in the dorsal horn of the spinal cord in an animal model of long-term diabetes. Diabetes was induced in Sprague-Dawley rats using 55 mg/kg streptozotocin, and expression of total CaMKII, the phosphorylated α-CaMKII isoform, and isolectin B4 was analyzed by immunohistochemical analysis in the dorsal horn of the spinal cord 6 and 12 months after diabetes induction. Results were compared with those for control rats of the same age. Increased expression of total CaMKII and its activated α isoform was seen in the dorsal horn of diabetic rats 6 months after diabetes induction. The increase in CaMKII fluorescence was restored to control values after 12 months. The expression of activated α-CaMKII 12 months after diabetes induction was most pronounced in laminae I-VI of the dorsal horn, not corresponding with the highest expression of isolectin B4 in laminae I-III. Increased expression of CaMKII in the dorsal horn during long-term diabetes could be involved in the development of neuropathic symptoms in diabetes. The expression pattern of CaMKII during long-term diabetes indicates that it affects the entire sensory input.