Changes in the expression of endocannabinoid system components in an experimental model of chemotherapy-induced peripheral neuropathic pain: Evaluation of sex-related differences.

Chemotherapy-induced neuropathic pain is a serious clinical problem and one of the major side effects in cancer treatment. The endocannabinoid system (ECS) plays a crucial role in regulating pain neurotransmission, and changes in the expression of different components of the ECS have been reported in experimental models of persistent pain. In addition, sex differences have been observed in ECS regulation and function. The aim of our study was to evaluate whether administration of oxaliplatin, a neurotoxic antineoplastic agent, induced changes in the expression of ECS components in peripheral and central stations of the pain pathway, and if those changes exhibited sexual dimorphism. Adult male and female rats were injected with oxaliplatin or saline, and mechanical and cold hypersensitivity and allodynia were evaluated using Von Frey and Choi Tests. The mRNA levels corresponding to cannabinoid receptors (CB1, CB2), cannabinoid-related receptors (GPR55, 5HT1A, TRPV1) and to the main enzymes involved in the synthesis (DAGL, DAGL, NAPE-PLD) and degradation (MGL, FAAH) of endocannabinoids were assessed in lumbar dorsal root ganglia (DRGs) and spinal cord by using real time RT-PCR. In addition, the levels of the main endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide (AEA), were evaluated using commercial ELISA kits. Oxaliplatin administration induced the development of mechanical and cold hypersensitivity and allodynia in male and female animals. Oxaliplatin also induced early and robust changes in the expression of several components of the ECS in DRGs. A marked upregulation of CB1, CB2, 5HT1A and TRPV1 was detected in both sexes. Interestingly, while DAGL mRNA levels remained unchanged, DAGL was downregulated in male and upregulated in female rats. Finally, MGL and NAPE-PLD showed increased levels only in male animals, while FAAH resulted upregulated in both sexes. In parallel, reduced 2-AG and AEA levels were detected in DRGs from male or female rats, respectively. In the lumbar spinal cord, only TRPV1 mRNA levels were found to be upregulated in both sexes. Our results reveal previously unreported changes in the expression of cannabinoid receptors, ligands and enzymes occurring mainly in the peripheral nervous system and displaying certain sexual dimorphism. These changes may contribute to the physiopathology of oxaliplatin-induced neuropathic pain in male and female rats. A better understanding of these dynamic changes will facilitate the development of mechanism- and sex-specific approaches to optimize the use of cannabinoid-based medicines for the treatment of chemotherapy-induced pain.

Sex-related differences in oxaliplatin-induced changes in the expression of transient receptor potential channels and their contribution to cold hypersensitivity.

Transient receptor potential (TRP) channels are involved in the development of oxaliplatin-induced neuropathic pain, a frequent and debilitating side effect of cancer therapy. Here we explored whether oxaliplatin-induced changes in the expression of TRP channels, as well as the development of pain-related behaviours, differed between male and female animals. Adult rats were injected with oxaliplatin or saline and mechanical and cold allodynia were evaluated using Von Frey and Choi Tests. The mRNA levels of TRPV1, TRPM8 and TRPA1 were assessed in lumbar ganglia and spinal cord by using real time RT-PCR. Oxaliplatin administration induced mechanical and cold hypersensitivity and allodynia in both sexes, with more severe responses to cold stimulation detected in females. Oxaliplatin also induced a significant increase in the expression of TRPV1, TRPM8 and TRPA1 in lumbar dorsal root ganglia. Interestingly, while TRPV1 and TRPA1 upregulation showed no sex difference, the increase in TRPM8 mRNA levels was more pronounced in female ganglia, correlating with the increased sensitivity to innocuous cold stimuli observed in females. TRPV1 and TRPM8 were also found to be upregulated in the spinal cord of animals of both sexes. Our results reveal previously undescribed changes in the expression of TRP channels occurring in peripheral ganglia and spinal cord of both male and female oxaliplatin-treated animals, with some of these changes exhibiting sex-related differences that could underlie the development of sex-specific patterns of pain-related behaviours.

Limitation of life-sustaining treatment in NICU: Physicians' beliefs and attitudes in the Buenos Aires region.

OBJECTIVE: To explore the ethical beliefs and attitudes of Argentinean neonatologists regarding limitation of life-sustaining treatment (LST) for very sick infants. METHODS: We used an anonymous questionnaire including direct questions and hypothetical clinical cases (inevitable demise and anticipated survival with severe long-term disability). Multivariable analysis was carried out to assess the relation between type of clinical case and physicians' LST attitudes. RESULTS: Overall, 315 neonatologists in 34 units in the Buenos Aires region participated (response rate 54%). Most responders would agree with decisions to start or continue LST. In both clinical cases, continuing current treatment with no therapeutic escalation was the only form of LST limitation acceptable to a substantial proportion (about 60%) of neonatologists. Agreement with LST limitation was slightly but significantly more likely when death was inevitable. CONCLUSION: Argentinean neonatologists showed a conservative attitude regarding LST limitation. Patient prognosis and options of non-treatment decision significantly influenced their choices.

Opinions of Argentinean neonatologists on the initiation of life-sustaining treatment in preterm infants.

BACKGROUND: In June 2014, the Argentinean Ministry of Health published guidelines for the management of neonates born at the limit of viability (≤25 weeks of gestation). We explored the opinion of neonatologists in Buenos Aires, Argentina, regarding the initiation of life-sustaining treatment (LST) in critically ill neonates, focusing on the effect of sociocultural factors on their opinion. METHODS: An anonymous survey was designed to explore the opinions of Argentinean neonatologists on whether or not to initiate LST in newborns born prematurely. Five hundred eighty neonatologists from 36 neonatal units were invited to participate, and 315 specialists from 34 neonatal units completed the survey (response rate 54%). The survey was conducted between June 2014 and February 2015. RESULTS: 9.5% (30/315) of the neonatologists answered they would begin LST on neonates born at 22 weeks, 42.5% (134/315) at 23 weeks, 37% (117/315) at 24 weeks, 7% (22/315) at 25 weeks, and 4% (12/315) at ≥26 weeks. Cumulatively then, 96% of participants stated they would start LST at 25 weeks of gestation or less. On multivariate analysis, a "transcendent" value of life and lack of consideration of the local legal framework for making medical decisions in the delivery room were statistically associated with an opinion in favor of initiation of LST in neonates born at the limit of viability. More than 50% of the Argentinean neonatologists surveyed answered they would initiate treatment at a gestational age of less than 23 weeks, despite the fact that the recommendations of the Argentinean Ministry of Health are to only give comfort care for these neonates. The opinion of most Argentinean neonatologists surveyed thus differs from that recommended by the guidelines of Argentina. CONCLUSION: The most frequent opinion of Argentinean neonatologists was to initiate LST in neonates at the limit of viability. Certain factors, in particular the sense of a transcendent meaning to life and lack of consideration of the local legal framework for making medical decisions in the delivery room, seem to influence the decision to start LST.

Long-lasting ameliorating effects of the oligodeoxynucleotide IMT504 on mechanical allodynia and hindpaw edema in rats with chronic hindpaw inflammation.

PURPOSE: Previously we showed that systemic administration of IMT504 prevents or ameliorates mechanical and thermal allodynia in rats with sciatic nerve crush. Here we analyzed if IMT504 is also effective in reducing mechanical allodynia and inflammation in rats undergoing hindpaw inflammation. MATERIALS AND METHODS: Male Sprague-Dawley rats received unilateral intraplantar injection of complete Freund́s adjuvant (CFA), and were grouped into: 1) untreated CFA, 2) vehicle-treated CFA, 3) IMT504-treated CFA (5 daily (5*) doses of 20, 2 or 0.2 mg/kg, or 3*2 mg/kg). Naïve groups were also included. Finally, early (immediately after intraplantar CFA) and late (7 days after intraplantar CFA) IMT504 treatment protocols were also tested. Hindpaw mechanical allodynia, dorsoventral thickness, edema and cellular infiltration of ipsilateral hindpaws were evaluated in all groups. RESULTS: Untreated CFA rats exhibited mechanical allodynia of quick onset (day 1) and long duration (7 weeks inclusive). Early and late treatments with 5*20 mg/kg IMT504 to CFA rats resulted in both quick and long-lasting antiallodynic effects, as compared to untreated CFA rats. This was also the case in CFA rats undergoing late IMT504 treatment at lower doses (3* and 5*2 mg/kg). Very low doses of IMT504 (5*0.2 mg/kg) only showed a mild improvement in withdrawal threshold, never reaching basal levels. Finally, rats treated with 3* or 5*2 mg/kg or 5*0.2 mg/kg exhibited significant decreases in dorsoventral thickness, edema, and inflammatory cell infiltration of the inflamed hindpaw. CONCLUSION: Early and late administration of IMT504 results in quick and long-lasting reductions in mechanical allodynia and hindpaw edema. While the mechanisms behind these effects remain to be established, data suggests that IMT504 administration could be a promising strategy in the control of inflammatory pain.

Medical students' palliative care education in a Latin American university: a three-year experience at Austral University in Buenos Aires, Argentina.

BACKGROUND: The School of Medicine of Austral University incorporated palliative care as an elective in undergraduate medicine curriculum during 2010. OBJECTIVE: We analyzed the experience and results after 3 years of teaching palliative care. We compared students who chose palliative care as an elective subject (PC Group) with students who did not (Non-PC Group). We focused on the experience of contact with palliative care patients and self-perceived attitudes. Additionally, the impact produced by palliative care education in knowledge, self-perceived attitudes, and comfort was evaluated. METHODS: All the students tested completed a questionnaire on their attitude when exposed to dying patients. Students in the PC Group completed an additional questionnaire to assess their level of knowledge and their self-perceived comfort when interacting with patients. RESULTS: We tested 146 students. All students in the PC Group and 95.2% in the Non-PC Group considered that specific death issues ought to be part of the curriculum. Some students indicated that they could be present in a mandatory course. Before taking their elective, students in the PC Group confirmed a lack of technical training to understand palliative care patients, as did those students in the Non-PC Group. After taking a palliative care elective students expressed an improvement in self-perceived attitudes toward suffering and there was a significant increase (p<0.0001-0.0045) in knowledge. They also expressed an improvement in comfort levels in evaluation and treatment of pain. More than 95% of the students in the PC Group rated the experience as valuable and perceived the content as not available elsewhere in their training. DISCUSSION/CONCLUSION: Our results show that palliative care education provides opportunities to improve attitudes not specific to this discipline: interprofessional collaboration, holistic care, patient-centered care, self-awareness, and humanism. We conclude that an exposure to palliative care improved student's perception about the complexities of dying patients and their care.

Progesterone prevents nerve injury-induced allodynia and spinal NMDA receptor upregulation in rats.

BACKGROUND: Peripheral nerve injury-evoked neuropathic pain still remains a therapeutic challenge. Recent studies support the notion that progesterone, a neuroactive steroid, may offer a promising perspective in pain modulation. OBJECTIVES: Evaluate the effect of progesterone administration on the development of neuropathic pain-associated allodynia and on the spinal expression of N-Methyl-D-Aspartate Receptor subunit 1 (NR1), its phosphorylated form (pNR1), and the gamma isoform of protein kinase C (PKCγ), all key players in the process of central sensitization, in animals subjected to a sciatic nerve constriction. METHODS: Male Sprague-Dawley rats were subjected to a sciatic nerve single ligature constriction and treated with daily subcutaneous injections of progesterone (16 mg/kg) or vehicle. The development of hindpaw mechanical and thermal allodynia was assessed using the von Frey and Choi tests, respectively. Twenty two days after injury, the number of neuronal profiles exhibiting NR1, pNR1, or PKCγ immunoreactivity was determined in the dorsal horn of the lumbar spinal cord. RESULTS: Injured animals receiving progesterone did not develop mechanical allodynia and showed a significantly lower number of painful responses to cold stimulation. In correlation with the observed attenuation of pain behaviors, progesterone administration significantly reduced the number of NR1, pNR1, and PKCγ immunoreactive neuronal profiles. CONCLUSIONS: Our results show that progesterone prevents allodynia in a rat model of sciatic nerve constriction and reinforce its role as a potential treatment for neuropathic pain.

Nursing education: the experience, attitudes, and impact of caring for dying patients by undergraduate Argentinian nursing students.

BACKGROUND: There is extensive research documenting serious deficiencies in undergraduate nursing education related to end-of-life care. Many nurses and nursing students have difficulties in dealing with death and report feeling anxious and unprepared to be with patients who are dying. In Argentina, education on palliative care, death, and dying has not been made part of the undergraduate nursing curriculum. METHODS: We performed a multicenter survey on undergraduate nursing education regarding the care of dying patients at eight schools of nursing in Buenos Aires, Argentina. We enrolled 680 students from first to fifth year. RESULTS: Students acknowledged interacting directly with dying patients. Attitudes toward dying patients were highly positive. Students of the fifth year expressed a less satisfying relationship with their patients than those from the first year; considered it as a less gratifying occupation, and also showed a greater preference for avoiding emotional involvement with those patients. DISCUSSION: Many of them described in short and very expressive phrases the emotional impact of their encounters with patients facing a life-threatening illness. Students perceived that this issue received more attention in humanistic rather than clinical subjects. Ninety-eight percent of students spontaneously demanded more training in end-of-life care. The interest and desire of undergraduate students to enhance their knowledge and experience in palliative care, demands more specific teaching contents. CONCLUSION: This suggests that in Argentina, improvements in undergraduate nursing training are urgently needed and would be well received by the students. It could be very useful to consider this topic as part of accreditation standards for nursing programs.

Rat bone marrow stromal cells and oligonucleotides in pain research.

In the last years, significant progress has been made in the medical treatment of pain. However, pathological pains, such us neuropathic pain, remain refractory to the currently available analgesics. Therefore, new therapeutic strategies are being evaluated. We have recently shown that both bone marrow stromal cells (MSCs) and the oligonucleotide IMT504 can prevent the development of mechanical and thermal allodynia when they are administered to rats subjected to a sciatic nerve crush. This chapter summarizes the laboratory techniques used to isolate and culture MSCs, administer both MSCs and IMT504, perform the nerve injury and determine mechanical and thermal sensitivities.

Teaching dying patient care in three universities in Argentina, Spain, and Italy.

We performed a multicenter comparative survey on undergraduate medical education regarding the care of dying patients at medical schools in three countries. We enrolled 380 first- and sixth-year medical students from Universidad Austral (Argentina), Universidad de Navarra (Spain), and Università Campus Biomedico di Roma (Italy). Answers to the questions were similar among the three universities. Students acknowledged interacting directly with dying patients in all cases. Attitudes toward dying patients were highly positive. Students spontaneously requested more training in end-of-life care. Some attitudes and wishes varied significantly from course to course. Students perceived that this issue received more attention in humanistic rather than in clinical subjects. Ninety-eight percent of students considered that death and helping patients to have a good death should be included in their training. Students' attitudes revealed high interest and poor training in end-of-life issues. Medical curricula should be improved to adequately address these issues.

Neuropathic pain and temporal expression of preprodynorphin, protein kinase C and N-methyl-D-aspartate receptor subunits after spinal cord injury.

Central neuropathic pain is refractory to conventional treatment and thus remains a therapeutic challenge. In this work, we used a well-recognized model of central neuropathic pain to evaluate time-dependent expression of preprodynorphin (ppD), protein kinase C gamma (PKCgamma) and NMDA receptor (NMDAR) subunits NR1, NR2A and NR2B, all critical players in nociceptive processing at the spinal level. Male Sprague-Dawley rats were subjected to spinal hemisection at T13 level and sham-operated rats were included as control animals. The development of hindpaw mechanical allodynia was assessed using the von Frey filaments test. Real time RT-PCR was employed to determine the relative mRNA levels of NMDAR subunits, ppD and PKCgamma in the dorsal spinal cord 1, 14 and 28 days after injury. Our results show that, coincident with the allodynic phase after injury, there was a strong up-regulation of the mRNAs coding for ppD, PKCgamma and NMDAR subunits in the dorsal spinal cord caudal to the injury site. The present study provides further evidence that these molecules are involved in the development/maintenance of central neuropathic pain and thus could be the target of therapeutic approaches.

Oligonucleotide IMT504 reduces neuropathic pain after peripheral nerve injury.

We have recently shown that the administration of bone marrow stromal cells (MSCs) prevents the development of mechanical and thermal allodynia in animals subjected to a sciatic nerve injury. Furthermore, exogenously administered MSCs have been shown to participate in the repair and regeneration of damaged tissues in a variety of animal models. However, some limitations of this therapeutic approach, basically related to the ex vivo cell manipulation procedure, have arisen. IMT504, the prototype of the PyNTTTTGT class of immunostimulatory oligonucleotides, stimulates MSC expansion both in vitro and in vivo. In this study, we evaluated the effect of IMT504 systemic administration on the development of mechanical and thermal allodynia in rats subjected to a sciatic nerve crush. Animals were treated with IMT504, MSCs or saline either immediately after performing the lesion or 4 days after it, and were evaluated using the von Frey and Choi tests at different times after injury. Control animals developed both mechanical and thermal allodynia. Animals receiving either IMT504 or MSCs immediately after injury did not develop mechanical allodynia and presented a significantly lower number of nociceptive responses to cold stimulation as compared to controls. Moreover, injury-induced allodynia was significantly reduced after IMT504 delayed treatment. Our results show that the administration of IMT504 reduces neuropathic pain-associated behaviors, suggesting that IMT504 could represent a possible therapeutic approach for the treatment of neuropathic pain.

Differential galanin upregulation in dorsal root ganglia and spinal cord after graded single ligature nerve constriction of the rat sciatic nerve.

Single ligature nerve constriction (SLNC) is a newly developed animal model for the study of neuropathic pain. SLNC of the rat sciatic nerve induces pain-related behaviors, as well as changes in the expression of neuropeptide tyrosine and the Y(1) receptor in lumbar dorsal root ganglia (DRGs) and spinal cord. In the present study, we have analyzed the expression of another neuropeptide, galanin, in lumbar DRGs and spinal cord after different degrees of constriction of the rat sciatic nerve. The nerve was ligated and reduced to 10-30, 40-80 or 90% of its original diameter (light, medium or strong SLNCs). At different times after injury (7, 14, 30, 60 days), lumbar 4 and 5 DRGs and the corresponding levels of the spinal cord were dissected out and processed for galanin-immunohistochemistry. In DRGs, SLNC induced a gradual increase in the number of galanin-immunoreactive (IR) neurons, in direct correlation with the degree of constriction. Thus, after light SLNC, a modest upregulation of galanin was observed, mainly in small-sized neurons. However, following medium or strong SLNCs, there was a more drastic increase in the number of galanin-IR neurons, involving also medium and large-sized cells. The highest numbers of galanin-IR neurons were detected 14 days after injury. In the dorsal horn of the spinal cord, medium and strong SLNCs induced a marked ipsilateral increase in galanin-like immunoreactivity in laminae I-II. These results show that galanin expression in DRGs and spinal cord is differentially regulated by different degrees of nerve constriction and further support its modulatory role on neuropathic pain.

Bone marrow stromal cells induce changes in pain behavior after sciatic nerve constriction.

Peripheral nerve injury, i.e. a single ligature nerve constriction (SLNC), triggers neuropathic pain. Bone marrow stromal cells (MSCs) have been observed to migrate to the injured tissues and mediate functional recovery following brain, spinal cord and peripheral nerve lesions. We have recently shown MSC selective migration to the ipsilateral lumbar (L3-6) dorsal root ganglia (DRGs) after a sciatic nerve SLNC. In this study, we have analyzed the thermal and mechanical sensitivities of animals subjected to a SLNC of the sciatic nerve and an ipsilateral intraganglionic MSC injection, using the von Frey and Choi tests. Control animals were subjected to the nerve lesion either alone or followed by the administration of phosphate-buffered saline (PBS) or bone marrow non-adherent mononuclear cells (BNMCs). All the animals were tested both before surgery and after 1, 3, 7, 14, 21, 28 and 56 days. Animals subjected to the sciatic nerve constriction developed ipsilateral mechanical and thermal allodynia already 3 days after the lesion. The allodynic responses were maintained even after 56 days. MSC administration prevented the generation of mechanical allodynia and reduced the number of allodynic responses to cold stimuli. On the contrary, the injection of either PBS or BNMCs could not counteract allodynia. These results suggest that MSCs may modulate pain generation after sciatic nerve constriction. The underlying mechanisms by which MSCs exert their actions on pain behavior need to be clarified.

Selective migration and engraftment of bone marrow mesenchymal stem cells in rat lumbar dorsal root ganglia after sciatic nerve constriction.

Bone marrow mesenchymal stem cells (MSCs) preferentially migrate to the injured hemisphere when administered intravenously to rats with traumatic or ischemic brain injuries. In this study, we have investigated the localization of MSCs injected into the lumbar-4 dorsal root ganglion (L4-DRG) of rats with a sciatic nerve single ligature nerve constriction (SLNC). MSCs were isolated by their adherence to plastic, cultured until confluence and labelled with Hoechst. Animals with a unilateral injection of MSCs were subjected to an ipsilateral, bilateral or contralateral SLNC. After 9 days, they were perfused and the lumbar DRGs were dissected out, cut in a cryostat and observed with a fluorescence microscope. Large numbers of Hoechst-positive cells were observed in the injected L4-DRG, distributed around primary afferent neurons, resembling the anatomical localization of glial cells. In animals with an ipsilateral SLNC, some cells were detected in the ipsilateral L3, L5 or L6-DRGs but not in the contralateral ganglia. In animals with a bilateral lesion, MSCs migrated to both the ipsilateral and contralateral DRGs whereas in animals with a contralateral ligature, MSCs migrated to the contralateral DRGs. These results suggest that MSCs preferentially engraft in DRGs hosting primary sensory neurons affected by a lesion of their peripheral branches. Further studies should be carried out in order to elucidate the molecular mechanisms involved in this migration and homing, in order to evaluate the possible use of MSCs as a new therapeutic strategy for the treatment of peripheral nerve neuropathies.