Livia Puljak

Dorsal root ganglion - a potential new therapeutic target for neuropathic pain.

A regional approach can protect our patients from often unacceptable adverse effects produced by systematically applied drugs. Regional therapeutic approaches, as well as interventions at the level of the peripheral nervous system and particularly the dorsal root ganglion (DRG), represent an alternative to the systemic application of therapeutic agents. This article provides an overview of DRG anatomical peculiarities, explains why the DRG is an important therapeutic target, and how animal models of targeted drug delivery can help us in the translation of basic research into clinical practice.

Characterization of ionotrophic purinergic receptors in hepatocytes

Ionotrophic purinergic (P2X) receptors function as receptor‐gated cation channels, where agonist binding leads to opening of a nonselective cation pore permeable to both Na+ and Ca2+. Based on evidence that extracellular adenosine 5′‐triphosphate (ATP) stimulates glucose release from liver, these studies evaluate whether P2X receptors are expressed by hepatocytes and contribute to ATP‐dependent calcium signaling and glucose release. Studies were performed in isolated hepatocytes from rats and mice and hepatoma cells from humans and rats. Transcripts and protein for both P2X4 and P2X7 were detectable, and immunohistochemistry of intact liver revealed P2X4 in the basolateral and canalicular domains. In whole cell patch clamp studies, exposure to the P2X4/P2X7 receptor agonist 2′3′‐O‐(4‐benzoyl‐benzoyl)‐adenosine 5′‐triphosphate (BzATP; 10 μM) caused a rapid increase in membrane Na+ conductance. Similarly, with Fluo‐3 fluorescence, BzATP induced an increase in intracellular [Ca2+]. P2X4 receptors are likely involved because the calcium response to BzATP was inhibited by Cu2+, and the P2X4 modulators Zn2+ and ivermectin (0.3‐3 μM) each increased intracellular [Ca2+]. Exposure to BzATP decreased cellular glycogen content; and P2X4 receptor messenger RNA increased in glycogen‐rich liver samples. Conclusion: These studies provide evidence that P2X4 receptors are functionally important in hepatocyte Na+ and Ca2+ transport, are regulated by extracellular ATP and divalent cation concentrations, and may constitute a mechanism for autocrine regulation of hepatic glycogen metabolism. (HEPATOLOGY 2007.)

Extracting data from figures with software was faster, with higher interrater reliability than manual extraction

OBJECTIVES To compare speed and accuracy of graphical data extraction using manual estimation and open source software. STUDY DESIGN AND SETTING Data points from eligible graphs/figures published in randomized controlled trials (RCTs) from 2009 to 2014 were extracted by two authors independently, both by manual estimation and with the Plot Digitizer, open source software. Corresponding authors of each RCT were contacted up to four times via e-mail to obtain exact numbers that were used to create graphs. Accuracy of each method was compared against the source data from which the original graphs were produced. RESULTS Software data extraction was significantly faster, reducing time for extraction for 47%. Percent agreement between the two raters was 51% for manual and 53.5% for software data extraction. Percent agreement between the raters and original data was 66% vs. 75% for the first rater and 69% vs. 73% for the second rater, for manual and software extraction, respectively. CONCLUSIONS Data extraction from figures should be conducted using software, whereas manual estimation should be avoided. Using software for data extraction of data presented only in figures is faster and enables higher interrater reliability.

Depression of Ca2+/calmodulin‐dependent protein kinase II in dorsal root ganglion neurons after spinal nerve ligation

The enzyme calcium/calmodulin‐dependent protein kinase II (CaMKII) is associated with memory and its α isoform is critical for development of activity‐induced synaptic changes. Therefore, we hypothesized that CaMKII is involved in altered function of dorsal root ganglion (DRG) neurons after neuronal injury. To test this hypothesis, Sprague–Dawley rats were made hyperalgesic by L5 and L6 spinal nerve ligation (SNL), and changes in total phosphorylated and unphosphorylated CaMKII (tCaMKII) and phosphorylated form of its α isoform (pCaMKIIα) were analyzed using immunochemistry in different subpopulations of DRG. SNL did not induce any changes in tCaMKII between experimental groups, while the overall percentage of pCaMKIIα‐positive neurons in injured L5 DRG SNL (24.8%) decreased significantly when compared to control (41.7%). SNL did not change the percentage of pCaMKIIα/N52 colabeled neurons but decreased the percentage of N52‐negative nonmyelinated neurons that expressed pCaMKIIα from 27% in control animals to 11% after axotomy. We also observed a significant decrease in the percentage of small nonpeptidergic neurons labeled with IB4 (37.6% in control vs. 4.0% in L5 SNL DRG), as well as a decrease in the percentage of pCaMKIIα/IB4 colabeled neurons in injured L5 DRGs (27% in control vs. 1% in L5 DRG of SNL group). Our results show that reduction in pCaMKIIα levels following peripheral injury is due to the loss of IB4‐positive neurons. These results indicate that diminished afferent activity after axotomy may lead to decreased phosphorylation of CaMKIIα. J. Comp. Neurol. 518:64–74, 2010.

Predictors of attrition and academic success of medical students: a 30-year retrospective study.

Aim To determine attrition and predictors of academic success among medical students at University of Split, Croatia. Methods We analysed academic records of 2054 students enrolled during 1979–2008 period. Results We found that 26% (533/2054) of enrolled students did not graduate. The most common reasons for attrition were ‘personal’ (36.4%), transfer to another medical school (35.6%), and dismissal due to unsatisfactory academic record (21.2%). Grade point average (GPA) and study duration of attrition students were significantly associated with parental education. There were 1126 graduates, 395 men and 731 women. Their average graduation GPA was 3.67±0.53 and study duration 7.6±2.44 years. During 5-year curriculum only 6.4% (42/654) of students graduated in time, and 55% (240/472) of students graduated in time after curriculum was extended to 6 years. Variables predicting whether a student will graduate or not were high school grades, entrance exam score and year of enrollment. Significant predictors of graduation grades were high school grades and entrance exam score. Entrance exam score predicted length of studying. Conclusion Preadmission academic qualifications and year of enrollment predict academic success in medical school. More attention should be devoted to high attrition.

Lidocaine Injection into the Rat Dorsal Root Ganglion Causes Neuroinflammation

BACKGROUND: Injury of a spinal nerve or dorsal root ganglion (DRG) during selective spinal nerve blocks is a potentially serious complication that has not been adequately investigated. Our hypothesis was that local anesthetic injection into these structures may result in an inflammatory response and hyperalgesia. METHODS: We evaluated inflammatory and behavioral responses after injection of 4 &mgr;L lidocaine or saline into the L5 spinal nerve or DRG of rats after partial laminectomy. Behavioral testing was performed before and after surgery to examine hyperalgesia in response to nociceptive mechanical stimulation of the foot. DRGs were harvested and stained, and rings of immunoreactive glial cells around neurons were counted. RESULTS: Animals demonstrated hyperalgesia on the ipsilateral paw up to 4 days after lidocaine injection into the DRG but not after injection into the spinal nerve. The number of glial fibrillary acid protein immunopositive glial cell rings, which represent activation of satellite cells, significantly increased in DRGs after injection of lidocaine into either the DRG or the spinal nerve. The number of glial fibrillary acid protein-positive cells in the lidocaine-injected group was significantly larger than in the saline-injected group. Sporadic OX-42 immunopositive cells, which represent activated microglia, were also seen in lidocaine-injected DRGs. Testing for Pan-T expression, which labels activated T lymphocytes, showed no positive cells. CONCLUSIONS: Lidocaine injection into the DRG may produce hyperalgesia, possibly due to activation of resident satellite glial cells. In a clinical setting, local anesthetic injection into the DRG should be avoided during selective spinal nerve blocks.

Expression of calcium/calmodulin-dependent protein kinase II and pain-related behavior in rat models of type 1 and type 2 diabetes

BACKGROUND:Abnormalities in peripheral nerves and dorsal root ganglia are noticed in the early stage of experimentally provoked diabetic neuropathy. Enzyme calcium/calmodulin-dependent protein kinase II (CaMKII) may have a modulating role in diabetic neuropathy because of its role in calcium homeostasis. METHODS:A model of type 1 diabetes mellitus (DM1) was induced with 55 mg/kg of the streptozotocin and for DM2 induction a combination of high-fat diet and low-dose streptozotocin (35 mg/kg) was used. Pain-related behavior was analyzed using thermal and mechanical stimuli. Two weeks and 2 months after induction of diabetes rats were euthanized, and the expression of CaMKII and its isoforms in the dorsal root ganglia were analyzed using immunofluorescence. RESULTS:Both types of diabetes were successfully induced, as confirmed by hyperglycemia. Increased pain-related behavior became evident in DM1 rats in 2 weeks after diabetes induction, but not in DM2 rats. The expression of total CaMKII and the phosphorylated &agr; isoform of CaMKII increased in DM1 animals concurrently with pain-related behavior. Expression of &agr;, &bgr;, &ggr;, and &dgr; isoforms in DM1 animals and expression of total CaMKII and all of its analyzed isoforms in DM2 animals remained unchanged. CONCLUSIONS:Our findings may indicate involvement of CaMKII in transmission of nociceptive input early in DM1, but not in DM2. CaMKII may be a suitable pharmacological target for diabetic neuropathy.

Targeted delivery of pharmacological agents into rat dorsal root ganglion

We sought an optimal method for targeted delivery into dorsal root ganglia (DRGs) for experimental studies, in terms of precision of delivery and avoidance of behavioral disturbances. We examined three approaches for injection into rat DRGs: percutaneous injection without surgical exposure, injection after deep exposure, and injection following deep exposure and partial laminectomy. Coomassie blue and Fast Blue were injected into DRGs for validation. At necropsy, the spread of Coomassie blue and Fast Blue was investigated under stereomicroscope and fluorescent microscope, respectively. We found that percutaneous approach did not provide any successful DRG injections. Deep exposure prior to intraganglionic injection provided variable results, but intraganglionic injection after deep exposure plus partial laminectomy was successful in 100% of attempts. Our subsequent skeletal analysis showed that the anatomical location of DRG is not compatible with successful DRG injection without surgical exposure. Neither of the methods using surgical exposure caused behavioral disturbances. Based on these results we conclude that partial laminectomy offers the most precise method of injecting DRG and does not produce behavioral evidence of nerve damage. Intraganglionic injection after deep exposure alone is less predictable, while percutaneous approaches only allow injection in the peripheral nerve.

Attenuation of pain-related behavior evoked by injury through blockade of neuropeptide Y Y2 receptor.

&NA; Neuropeptide Y (NPY) has an important but still insufficiently defined role in pain modulation. We therefore examined the ability of NPY to modulate experimentally induced neuropathic pain by injecting it directly into dorsal root ganglion (DRG) immediately following spinal nerve ligation (SNL) injury. We have found that this application exacerbates pain‐related behavior induced by SNL in a modality‐specific fashion. When saline was injected after SNL, the expected increase in hyperalgesia responses to needle stimulation was present on the 8th postoperative day. When we injected NPY, hyperalgesic responses were increased in a manner similar to the SNL/saline group. To characterize NPY action, specific Y1 and Y2 antagonists were also delivered directly to DRG, which revealed that behavioral actions of NPY were abolished by Y2 receptor antagonist. We tested whether NPY effects were the result of its role in immunity by immunohistochemical staining for glial fibrillary acidic protein, in order to identify activation of DRG satellite cells and dorsal horn astrocytes. Exacerbation of pain‐related behavior following NPY injection was accompanied by astrocyte activation in ipsilateral dorsal horn and with satellite cells activation in the DRG proximal to injury. This activation was reduced following Y2 receptor antagonist application. These findings indicate an important link between pain‐related behavior and neuroimmune activation by NPY through its Y2 receptor. Prevention of early changes in dorsal root neurons by neuropeptide Y Y2 receptor can reduce behavioral consequences of spinal nerve ligation.

Increased Phosphoenolpyruvate Carboxykinase Gene Expression and Steatosis during Hepatitis C Virus Subgenome Replication ROLE OF NONSTRUCTURAL COMPONENT 5A AND CCAAT/ENHANCER-BINDING PROTEIN β

Background: Chronic hepatitis C virus (HCV) infection increases the risk of type 2 diabetes and hepatic steatosis. Results: Phosphoenolpyruvate carboxykinase (PEPCK) and associated transcription factors are up-regulated in HCV-infected Huh.8 cells. Conclusion: Increased CCAAT/enhancer-binding protein β (C/EBPβ) and nonstructural component 5A (NS5A) are essential components for increased gluconeogenesis. Significance: NS5A and C/EBPβ may possibly be considered as a new pharmacological target during HCV infection. Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated transcription factors are dramatically up-regulated in Huh.8 cells, which stably express an HCV subgenome replicon. HCV increased activation of cAMP response element-binding protein (CREB), CCAAT/enhancer-binding protein (C/EBPβ), forkhead box protein O1 (FOXO1), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and involved activation of the cAMP response element in the PEPCK promoter. Infection with dominant-negative CREB or C/EBPβ-shRNA significantly reduced or normalized PEPCK expression, with no change in PGC-1α or FOXO1 levels. Notably, expression of HCV nonstructural component NS5A in Huh7 or primary hepatocytes stimulated PEPCK gene expression and glucose output in HepG2 cells, whereas a deletion in NS5A reduced PEPCK expression and lowered cellular lipids but was without effect on insulin resistance, as demonstrated by the inability of insulin to stimulate mobilization of a pool of insulin-responsive vesicles to the plasma membrane. HCV-replicating cells demonstrated increases in cellular lipids with insulin resistance at the level of the insulin receptor, increased insulin receptor substrate 1 (Ser-312), and decreased Akt (Ser-473) activation in response to insulin. C/EBPβ-RNAi normalized lipogenic genes sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor γ, and liver X receptor α but was unable to reduce accumulation of triglycerides in Huh.8 cells or reverse the increase in ApoB expression, suggesting a role for increased lipid retention in steatotic hepatocytes. Collectively, these data reveal an important role of NS5A, C/EBPβ, and pCREB in promoting HCV-induced gluconeogenic gene expression and suggest that increased C/EBPβ and NS5A may be essential components leading to increased gluconeogenesis associated with HCV infection.