Clinical Outcome of Sacral Chordoma Patients Treated with Pencil Beam Scanning Proton Therapy.

AIMS: Sacral chordomas are locally aggressive, radio-resistant tumours. Proton therapy has the potential to deliver high radiation doses, which may improve the therapeutic ratio when compared with conventional radiotherapy. We assessed tumour control and radiation-induced toxicity in a cohort of sacral chordoma patients treated with definitive or postoperative pencil beam scanning proton therapy. METHODS AND MATERIALS: Sixty patients with histologically proven sacral chordoma treated between November 1997 and October 2018 at the Paul Scherrer Institute with postoperative (n = 50) or definitive proton therapy (n = 10) were retrospectively analysed. Only 10 (17%) patients received combined photon radiotherapy and proton therapy. Survival rates were calculated using the Kaplan-Meier actuarial method. The Log-rank test was used to compare different functions for local control, freedom from distant recurrence and overall survival. Acute and late toxicity were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) v5.0. RESULTS: The median follow-up was 48 months (range 4-186). Local recurrence occurred in 20 (33%) patients. The 4-year local control, freedom from distant recurrence and overall survival rates were 77%, 89% and 85%, respectively. On univariate analysis, subtotal resection/biopsy (P = 0.02), tumour extension restricted to bone (P = 0.01) and gross tumour volume >130 ml (P = 0.04) were significant predictors for local recurrence. On multivariate analysis, tumour extension restricted to bone (P = 0.004) and gross total resection (P = 0.02) remained independent favourable prognostic factors for local recurrence. Twenty-four (40%), 28 (47%) and eight (11%) patients experienced acute grade 1, 2 and 3 toxicities, respectively. The 4-year late toxicity-free survival was 91%. Two patients developed secondary malignancies to the bladder 3-7 years after proton therapy. CONCLUSIONS: Our data indicate that pencil beam scanning proton therapy for sacral chordomas is both safe and effective. Gross total resection, tumour volume <130 ml and tumour restricted to the bone are favourable prognostic factors for local tumour control.

[Malignant tumors of the oral cavity]. / Tumoren der Mundhöhle.

CLINICAL/METHODICAL ISSUE: Oral cavity malignancies are the most common tumors in the field of ear, nose and throat medicine or otorhinolaryngology worldwide. It comprises a heterogeneous group of tumors, the knowledge of which is necessary to meet the different requirements of diagnostics and therapy. STANDARD RADIOLOGICAL METHODS: Computed tomography (CT), magnetic resonance imaging (MRI), sonography (US), nuclear medical procedures (NUK). PERFORMANCE: The above-mentioned diagnostics are used in a complementary manner. ACHIEVEMENTS: Early diagnosis of the tumor improves staging and thus the patient's therapy and prognosis. PRACTICAL RECOMMENDATIONS: The radiologist plays an important role in the interdisciplinary treatment of malignant tumors of the oral cavity. Despite great progress in radiotherapy, oncology and immunotherapy, surgery still plays an important role in the treatment of malignant diseases of the oral cavity.

Outcomes, Prognostic Factors and Salvage Treatment for Recurrent Chordoma After Pencil Beam Scanning Proton Therapy at the Paul Scherrer Institute.

AIMS: The outcome of chordoma patients with local or distant failure after proton therapy is not well established. We assessed the disease-specific (DSS) and overall survival of patients recurring after proton therapy and evaluated the prognostic factors affecting DSS. MATERIALS AND METHODS: A retrospective analysis was carried out of 71 recurring skull base (n = 36) and extracranial (n = 35) chordoma patients who received adjuvant proton therapy at initial presentation (n = 42; 59%) or after post-surgical recurrence (n = 29; 41%). The median proton therapy dose delivered was 74 GyRBE (range 62-76). The mean age was 55 ± 14.2 years and the male/female ratio was about one. RESULTS: The median time to first failure after proton therapy was 30.8 months (range 3-152). Most patients (n = 59; 83%) presented with locoregional failure only. There were only 12 (17%) distant failures, either with (n = 5) or without (n = 7) synchronous local failure. Eight patients (11%) received no salvage therapy for their treatment failure after proton therapy. Salvage treatments after proton therapy failure included surgery, systemic therapy and additional radiotherapy in 45 (63%), 20 (28%) and eight (11%) patients, respectively. Fifty-three patients (75%) died, most often from disease progression (47 of 53 patients; 89%). The median DSS and overall survival after failure was 3.9 (95% confidence interval 3.1-5.1) and 3.4 (95% confidence interval 2.5-4.4) years, respectively. On multivariate analysis, extracranial location and late failure (≥31 months after proton therapy) were independent favourable prognostic factors for DSS. CONCLUSION: The survival of chordoma patients after a treatment failure following proton therapy is poor, particularly for patients who relapse early or recur in the skull base. Although salvage treatment is administered to most patients with uncontrolled disease, they will ultimately die as a result of disease progression in most cases.

Pencil Beam Scanning Proton Therapy for Paediatric Neuroblastoma with Motion Mitigation Strategy for Moving Target Volumes.

AIMS: More efforts are required to minimise late radiation side-effects for paediatric patients. Pencil beam scanning proton beam therapy (PBS-PT) allows increased sparing of normal tissues while maintaining conformality, but is prone to dose degradation from interplay effects due to respiratory motion. We report our clinical experience of motion mitigation with volumetric rescanning (vRSC) and outcomes of children with neuroblastoma. MATERIALS AND METHODS: Nineteen patients with high-risk (n = 16) and intermediate-risk (n = 3) neuroblastoma received PBS-PT. The median age at PBS-PT was 3.5 years (range 1.2-8.6) and the median PBS-PT dose was 21 Gy (relative biological effectiveness). Most children (89%) were treated under general anaesthesia. Seven patients (37%) underwent four-dimensional computed tomography for motion assessment and were treated with vRSC for motion mitigation. RESULTS: The mean result of maximum organ motion was 2.7 mm (cranial-caudal), 1.2 mm (left-right), 1.0 mm (anterior-posterior). Four anaesthetised children (21%) showing <5 mm motion had four-dimensional dose calculations (4DDC) to guide the number of vRSC. The mean deterioration or improvement to the planning target volume covered by 95% of the prescribed dose compared with static three-dimensional plans were: 4DDC no vRSC, -0.6%; 2 vRSC, +0.3%; 4 vRSC, +0.3%; and 8 vRSC, +0.1%. With a median follow-up of 14.9 months (range 2.7-49.0) there were no local recurrences. The 2-year overall survival was 94% and distant progression-free survival was 76%. Acute grade 2-4 toxicity was 11%. During the limited follow-up time, no late toxicities were observed. CONCLUSIONS: The early outcomes of mainly high-risk patients with neuroblastoma treated with PBS-PT were excellent. With a subset of our cohort undergoing PBS-PT with vRSC we have shown that it is logistically feasible and safe. The clinical relevance of vRSC is debatable in anaesthetised children with small pre-PBS-PT motion of <5 mm.

Patterns of care in recurrent glioblastoma in Switzerland: a multicentre national approach based on diagnostic nodes.

Despite moderate improvements in outcome of glioblastoma after first-line treatment with chemoradiation recent clinical trials failed to improve the prognosis of recurrent glioblastoma. In the absence of a standard of care we aimed to investigate institutional treatment strategies to identify similarities and differences in the pattern of care for recurrent glioblastoma. We investigated re-treatment criteria and therapeutic pathways for recurrent glioblastoma of eight neuro-oncology centres in Switzerland having an established multidisciplinary tumour-board conference. Decision algorithms, differences and consensus were analysed using the objective consensus methodology. A total of 16 different treatment recommendations were identified based on combinations of eight different decision criteria. The set of criteria implemented as well as the set of treatments offered was different in each centre. For specific situations, up to 6 different treatment recommendations were provided by the eight centres. The only wide-range consensus identified was to offer best supportive care to unfit patients. A majority recommendation was identified for non-operable large early recurrence with unmethylated MGMT promoter status in the fit patients: here bevacizumab was offered. In fit patients with late recurrent non-operable MGMT promoter methylated glioblastoma temozolomide was recommended by most. No other majority recommendations were present. In the absence of strong evidence we identified few consensus recommendations in the treatment of recurrent glioblastoma. This contrasts the limited availability of single drugs and treatment modalities. Clinical situations of greatest heterogeneity may be suitable to be addressed in clinical trials and second opinion referrals are likely to yield diverging recommendations.

Effect of rMnSOD on survival signaling in pediatric high risk T-cell acute lymphoblastic leukaemia.

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that defends against oxidative damage due to reactive oxygen species (ROS). A new isoform of MnSOD with cytotoxic activity was recently discovered in liposarcoma cells. Here, we tested the effectiveness of a recombinant form of this isoform (rMnSOD) on leukemic T cells, Jurkat cells, and lymphocytes. Our results confirm that leukemic T cells can internalize rMnSOD and that rMnSOD causes apoptosis of 99% of leukemic cells without showing toxic effects on healthy cells. Using light and electron microscopy, we determined that an rMnSOD concentration of 0.067 µM most effective on apoptosis induction. Western blot analysis showed that treatment with 0.067 µM rMnSOD resulted in high expression of the pro-apoptotic protein Bax and low expression of the anti-apoptotic protein Bcl-2 in leukemia cells. Concerning signal transduction pathway no influence was observed after treatment except for Jurkat cells showing a slightly decreased expression of ERK phosphorylation. These results suggest that rMnSOD may be an effective and non-toxic treatment option for T-cell leukemia.

Adjuvant therapy after resection of brain metastases. Frameless image-guided LINAC-based radiosurgery and stereotactic hypofractionated radiotherapy.

BACKGROUND: Tumor bed stereotactic radiosurgery (SRS) after resection of brain metastases is a new strategy to delay or avoid whole-brain irradiation (WBRT) and its associated toxicities. This retrospective study analyzes results of frameless image-guided linear accelerator (LINAC)-based SRS and stereotactic hypofractionated radiotherapy (SHRT) as adjuvant treatment without WBRT. MATERIALS AND METHODS: Between March 2009 and February 2012, 44 resection cavities in 42 patients were treated with SRS (23 cavities) or SHRT (21 cavities). All treatments were delivered using a stereotactic LINAC. All cavities were expanded by ≥ 2 mm in all directions to create the clinical target volume (CTV). RESULTS: The median planning target volume (PTV) for SRS was 11.1 cm(3). The median dose prescribed to the PTV margin for SRS was 17 Gy. Median PTV for SHRT was 22.3 cm(3). The fractionation schemes applied were: 4 fractions of 6 Gy (5 patients), 6 fractions of 4 Gy (6 patients) and 10 fractions of 4 Gy (10 patients). Median follow-up was 9.6 months. Local control (LC) rates after 6 and 12 months were 91 and 77 %, respectively. No statistically significant differences in LC rates between SRS and SHRT treatments were observed. Distant brain control (DBC) rates at 6 and 12 months were 61 and 33 %, respectively. Overall survival (OS) at 6 and 12 months was 87 and 63.5 %, respectively, with a median OS of 15.9 months. One patient treated by SRS showed symptoms of radionecrosis, which was confirmed histologically. CONCLUSION: Frameless image-guided LINAC-based adjuvant SRS and SHRT are effective and well tolerated local treatment strategies after resection of brain metastases in patients with oligometastatic disease.

Evaluation of negative pressure vacuum-assisted system in acute and chronic wounds closure: our experience.

Negative-pressure therapy or vacuum-assisted closure (VAC) has been used in clinical applications since the 1940's and has increased in popularity over the past decade. This dressing technique consists of an open cell foam dressing put into the wound cavity, a vacuum pump produces a negative pressure and an adhesive drape. A controlled sub atmospheric pressure from 75 to 150 mmHg is applied. The vacuum-assisted closure has been applied by many clinicians to chronic wounds in humans; however it cannot be used as a replacement for surgical debridement. The initial treatment for every contaminated wound should be the necrosectomy. The VAC therapy has a complementary function and the range of its indications includes pressure sores, stasis ulcers, chronic wounds such as diabetic foot ulcers, post traumatic and post operative wounds, infected wounds such as necrotizing fasciitis or sternal wounds, soft-tissue injuries, bone exposed injuries, abdominal open wounds and for securing a skin graft. We describe our experience with the VAC dressing used to manage acute and chronic wounds in a series of 135 patients, with excellent results together with satisfaction of the patients.

[Indications and results of stereotactic radiosurgery with LINAC for the treatment of acoustic neuromas: preliminary results]. / Indications et résultats du traitement des neurinomes de l'acoustique par radiothérapie stéréotaxique avec LINAC: résultats préliminaires.

INTRODUCTION: Radiosurgery is presently becoming an alternative to microsurgical resection of acoustic neuromas. The interest of radiosurgery consists in its lower morbidity compared to surgery and likely in similar rates of long-term tumor control. The goal of our study was to assess the clinical outcome (hearing preservation and neurological complications) as well as tumor control after low-dose radiosurgery for unilateral acoustic neuromas. MATERIAL AND METHODS: Since April 2002, 22 patients with untreated acoustic neuromas underwent stereotactic radiosurgery using a linear accelerator (LINAC) and a micromultileaf collimator (mMLC, Brain Lab) at a low-dose of 12 Gy. The average age was 56.4 years (range 29-73 years). The treatment volume was 0.03 to 6.04 cm(3) (median 1.85 cm(3)). The median follow-up period was 18 months (range 6-36 months). RESULTS: No morbidity was observed during the treatment. Preservation of a serviceable hearing (classes I and II according to the Gardner-Robertson scale) was achieved in 10 of 14 patients (71%). Radiological tumor growth control was obtained in all patients (100%). Trigeminal neuropathy was observed in two patients. One of these patients also experienced a slight facial weakness. CONCLUSIONS: Low dose radiosurgery provides a low rate of post-therapeutic morbidity and yields the preservation of a serviceable hearing in 70% of cases. Tumor control is observed in all patients, but a longer follow-up period is needed to confirm the stability of the tumor size.

Use of transgenic mice in acid-base balance studies.

The kidney is essential in maintaining body acid-base status. Recently, the use of transgenic mice has largely contributed to the understanding of the mechanisms involved. Important issues have been addressed in terms of the function of proteins or their regulation. In the proximal tubule, the role of Na+/HCO3-cotransport has been established, although further studies are needed to understand how its mutations lead to renal disease. Na+/H+ exchange has also been extensively studied, and its role in diuretic and natriuretic responses following an increase in blood pressure has been elucidated. The interaction of other transport proteins, such as the Na+/phosphate cotransporter NaPi II-a, with the Na+/H+ exchanger has also been investigated. In the medullary thick ascending limb of Henle's loop (MTAL), a role for NHE1 in transepithelial HCO3- absorption has been demonstrated: basolateral NHE1 controls the function of apical NHE3. As for the distal nephron, the majority of observations suggest that the regulation of H+-ATPase activity in response to acid-base status is mediated by the trafficking of pumps or pump sub-units, especially for the a4 subunit, rather than changes in subunit expression levels. Furthermore, the function of pendrin, a chloride/anion exchanger, has been assessed in response to changes in acid-base status. Important results have been obtained regarding the regulation of proximal tubule transport by several mechanisms, such as microvilli changes and the inducible and endothelial isoform of nitric oxide synthase (NOS). Finally, the interaction of chloride channels and potassium-chloride cotransporter with proton secretion has been evaluated. These findings highlight the importance of knockout animal models in studying kidney regulation of acid-base balance.

The origin of brain metastases in patients with an undiagnosed primary tumour.

BACKGROUND: In patients presenting brain metastases as the first manifestation of a previously undiagnosed primary tumour (UDP) histopathological confirmation of the diagnosis can be obtained by either direct surgical sampling of the brain lesion or paraclinical search for an accessible primary tumour. The sequence of the diagnostic work-up and the timing of an eventual neurosurgical intervention are a matter of debate and are mainly influenced by the distribution of primary tumours in UDP patients. The aim of this study was to verify the hypothesis that the distribution of primary tumours differs between UDP patients and the rest of the patients with brain metastases (DP), and to propose a diagnostic work-up specifically tailored to the UDP population. METHODS: Retrospective study on 342 patients admitted to the Lausanne University hospital between 1983 and 1998 with the diagnosis of cerebral metastases. FINDINGS: UDP patients represented 36% of the whole group. Primary tumour location was significantly different between the two groups (p=0.001). Although the lung was the most frequent primary tumour location in both groups (UDP: 60%, DP: 43%), in UDP 14% only of the primaries were found outside of the lung and as much as 26% remained unknown despite thorough investigations. CONCLUSIONS: Our study confirmed the hypothesis that the relative frequency of primary tumours differs between DP and UDP patients. This difference therefore mandates a diagnostic strategy specifically tailored for UDP patients: if a radiological lung investigation clearly remains the best initial step in the work-up of these patients, extensive paraclinical investigations without a clear clinical suspicion should probably not be undertaken if this first survey fails to disclose the primary tumour as only 14% of the patients will actually benefit from it. In this situation, a neurosurgical procedure should probably be considered the most appropriate next step to be taken in order to provide a definitive diagnosis without unnecessary delays.

Morphological and biochemical characterization of mitochondria in Torpedo red blood cells.

A study is presented on the morphology and respiratory functions of mitochondria from Torpedo marmorata red blood cells. In vivo staining of red blood cells and transmission electron microscopy showed the existence of a considerable number of vital and orthodox mitochondria which decreased from young erythroblasts to mature erythrocytes from 60-50 to 30-20 per cell. In erythrocytes mitochondria exhibited a canonical, functional respiratory chain. The content and activity of cytochromes in erythrocytes were, however, significantly lower as compared to mammalian tissues.

Beta-radiation for coronary in-stent restenosis.

To determine the feasibility and safety of an intracoronary beta-radiation device in preventing the recurrence of in-stent restenosis (ISR) after successful angioplasty, we studied 37 patients treated with beta-radiation (30-mm strontium-90 source) after angioplasty. The mean reference diameter was 2.9 +/- 0.5 mm, and 62% of lesions were diffuse, including four total occlusions. Beta-radiation was successfully delivered in 36 of 37 (97%) cases. Over the course of 7.1 +/- 4.5 mo follow-up, there were no myocardial infarctions and three deaths: one from preexisting malignancy, one from progressive cardiac failure, and one from sudden cardiac death. Target vessel revascularization (TVR) was performed in seven of 36 (19%) patients. Thirty patients underwent angiography at 6 mo; three (10%) experienced restenosis (diameter stenosis > 50%) at the target site, four (13%) had edge stenoses, and two (7%) had late (> 1 mo) thrombotic occlusions. Beta-radiation for ISR is associated with encouragingly low rates of target lesion restenosis and TVR. Further improvements are needed to solve the limitations of the edge effect and late occlusion.

Co-induction of nitric oxide synthase, bcl-2 and growth-associated protein-43 in spinal motoneurons during axon regeneration in the lizard tail.

In lizards, tail loss transects spinal nerves and the cut axons elongate in the regrowing tail, providing a natural paradigm of robust regenerative response of injured spinal motoneurons. We previously ascertained that these events involve nitric oxide synthase induction in the axotomized motoneurons, suggesting a correlation of this enzyme with regeneration-associated gene expression. Here we investigated, in lizards, whether the cell death repressor Bcl-2 protein and growth-associated protein-43 (GAP-43) were also induced in motoneurons that innervate the regenerated tail in the first month post-caudotomy. Single and multiple immunocytochemical techniques, and quantitative image analysis, were performed. Nitric oxide synthase, GAP-43 or Bcl-2 immunoreactivity was very low or absent in spinal motoneurons of control lizards with intact tail. Nitric oxide synthase and GAP-43 were induced during the first month post-caudotomy in more than 75% of motoneurons which innnervate the regenerate. Bcl-2 was induced in approximately 95% of these motoneurons at five and 15days, and in about 35% at one month. The intensity of Bcl-2 and GAP-43 immunostaining peaked at five days, and nitric oxide synthase at 15days; immunoreactivity to these proteins was still significantly high at one month. Immunofluorescence revealed co-localization of nitric oxide synthase, GAP-43 and Bcl-2 in the vast majority of motoneurons at five and 15days post-caudotomy. These findings demonstrate that co-induction of nitric oxide synthase, Bcl-2 and GAP-43 may be part of the molecular repertoire of injured motoneurons committed to survival and axon regeneration, and strongly favor a role of nitric oxide synthase in motoneuron plasticity.

Inhibition of neutral endopeptidase potentiates the effects of atrial natriuretic peptide on acute cyclosporin-induced nephrotoxicity.

BACKGROUND: The use of cyclosporin A (CyA) is limited by its significant nephrotoxicity. Atrial natriuretic peptide (ANP) has been shown to ameliorate the reduction in glomerular filtration rate (GFR) induced by CyA, but its effect is transient. One explanation may be the rapid breakdown of this hormone by neutral endopeptidase (NEP) which is highly active in the kidney. In the present study, we examined the effect of the NEP inhibitor thiorphan on the acute fall in GFR induced by CyA. METHODS: After a first set of experiments to investigate the renal hemodynamic effects of CyA (20 mg.kg(-1), i.v. bolus), we studied four additional conditions where acute CyA treatment was followed by the administration of: (2) ANP alone (10 microg.kg(-1) i.v. as bolus and a maintenance infusion of 1 microg. kg(-1).min(-1)); (3) thiorphan alone (5 mg.kg(-1) i.v. as bolus and a maintenance infusion of 0.5 mg.kg(-1). min(-1)); (4) ANP plus thiorphan (as in 2 and 3), and (5) an infusion of 0.9% saline, increased from 1.2 to 3 ml.h(-1). The GFR was measured as the clearance of (3)H-methoxyinulin (ml.min(-1).100 g(-1) body weight). RESULTS: The data show: (1) the GFR fell from 1.06 +/- 0.15 to 0.59 +/- 0.09 ml.min(-1).100 g(-1) (p < 0.01) 60 min after CyA and remained depressed for at least 2 h; (2) ANP caused a marked initial rise in GFR from 0.49 +/- 0.07 to 1.23 +/- 0.18 ml.min(-1).100 g(-1) (p < 0.005 vs. CyA) which declined rapidly to the value seen after CyA injection alone, despite continuing ANP infusion; (3) thiorphan caused a modest, but significant increase in GFR within 15 min from 0.48 +/- 0.04 to 0.69 +/- 0.10 ml.min(-1).100 g(-1) (p < 0.05 vs. CyA) which was sustained during infusion and for at least 30 min after stopping infusion; (4) ANP plus thiorphan produced a marked increase in GFR from 0.58 +/- 0.09 to 1.39 +/- 0.44 ml.min(-1).100 g(-1) (p < 0.05 vs. CyA) which then decreased, but remained above the post-CyA injection value, until infusion of both drugs ended; (5) more than doubling the saline infusion rate per se had no significant effect on the GFR response to CyA. The blood pressure decreased significantly during ANP infusion, but more so when combined with thiorphan. CONCLUSION: These data indicate that the inhibition of NEP by thiorphan is able to ameliorate partially the reduction in GFR induced by CyA and to enhance, and prolong, the vasodilator and diuretic effects of ANP.

First-line chemotherapy with local treatment can prevent external-beam irradiation and enucleation in low-stage intraocular retinoblastoma.

PURPOSE: To evaluate the efficacy of first-line chemotherapy (CT) in preventing external-beam radiotherapy (EBR) and/or enucleation in patients with retinoblastoma (Rbl). PATIENTS AND METHODS: Twenty-four patients with newly diagnosed unilateral or bilateral Rbl received CT associated with local treatment (LT). Two to five courses of etoposide and carboplatin were administered at 3- to 4-week intervals, depending on tumor response, and were completed each time by LT. RESULTS: Tumor response was observed in all eyes. Twenty-one of 24 patients showed a complete response (CR) that persisted at a median follow-up (FU) of 31 months (range, 4 to 41 months). Among the three patients who relapsed, two were lost to FU and one died of progressive disease. CR was achieved by CT and LT alone in 15 (71.4%) of 21 patients with less advanced disease (groups I to III). Six other patients with advanced disease (groups IV and V) experienced treatment failure and needed salvage treatment by EBR and/or enucleation. The difference between the two patient groups with regard to disease stage was statistically significant (P <.0001). EBR could be avoided in 13 (68.4%) of 19 patients, who presented with groups I to III (15 eyes) and group V (one eye) disease, whereas enucleation could be avoided in only two (40%) of five. CONCLUSION: CT combined with intensive LT is effective in patients with groups I to III Rbl, permitting the avoidance of EBR in the majority of these young children and, thus, reducing the risk of long-term sequelae. This is in contrast with the disappointing results for patients with groups IV and V Rbl, in whom EBR and/or enucleation was needed.

Plastic changes and nitric oxide synthase induction in neurons which innervate the regenerated tail of the lizard Gekko gecko. II. The response of dorsal root ganglion cells to tail amputation and regeneration.

The lizard tail regenerates after amputation, which severs the spinal cord and spinal nerves. Dorsal root ganglia (DRGs) do not regenerate in the regrowing tail, which is innervated by DRGs rostral to the amputation. With Nissl staining, NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry, we investigated NOS expression and its relationship with structural changes in DRG neurons of caudotomized lizards. First, by horseradish peroxidase retrograde tracing we here provided evidence that the sensory innervation of the regenerated tail derives only from the three pairs of DRGs rostral to the amputation plane. These ganglia were then analyzed in control animals with original intact tail, at 5, 15 and 30 days after caudotomy, and at 8 months in lizards with mature regenerates. Caudotomy elicited in DRG neurons marked hypertrophy that persisted after tail regeneration. In control ganglia, most neurons were lightly NADPH-diaphorase-positive, a few were unstained or intensely stained. Tail transection elicited marked staining up-regulation, and an increase in the proportion of intensely positive neurons. The staining intensity peaked in DRG neurons at 15 days and was still significantly increased in respect to controls several months after complete tail regeneration. NOS immunoreactivity in DRGs matched the histochemical findings. NADPH-diaphorase positivity was also enhanced in the dorsal horn superficial laminae of the corresponding spinal segments. We demonstrate that transection of the lizard spinal nerves, provoked by tail loss, elicits in the axotomized primary sensory neurons marked NOS enhancement, which accompanies axon elongation in the regrowing tail and persists after the end of this process.

Plastic changes and nitric oxide synthase induction in neurons that innervate the regenerated tail of the lizard Gekko gecko: I. Response of spinal motoneurons to tail amputation and regeneration.

The lizard tail regenerates after autotomy or amputation. After horseradish peroxidase injections in the regenerate, motoneurons were retrogradely labeled only in the three spinal segments rostral to the amputation, whose spinal nerves are severed by tail loss. The changes in these motoneurons, compared to those of lizards with original intact tails, were investigated 5, 15, and 30 days after caudotomy and at 8 months in lizards with mature regenerates. Morphometric analysis of Nissl-stained motoneurons rostral to the amputation revealed marked hypertrophy, peaking at 15 days, when chromatolysis and nuclear eccentricity were also evident; motoneuron perikarya remained significantly larger than in controls after tail regeneration. The dUTP nick-end labeling (TUNEL) stain for apoptotic neurons did not reveal labeled cells in the spinal cord 5 and 15 days after caudotomy. Nitric oxide synthase (NOS) expression was studied with nicotinamide adenine-dinucleotide phosphate (NADPH)-diaphorase histochemistry and evaluated quantitatively with densitometry. A few caudal spinal motoneurons were lightly stained in lizards with intact tails. Induction of NADPH-diaphorase positivity was evident in the vast majority of these cells 5 days after caudotomy and was very marked at 15 and 30 days, during tail regrowth. These data were confirmed by neuronal NOS immunohistochemistry. After tail regeneration, histochemical positivity was markedly down-regulated in the tail spinal motoneurons but persisted in the majority of these cells. The findings show that in the lizard caudotomy elicits in axotomized caudal spinal motoneurons NOS induction associated with plasticity phenomena and in particular with vigorous regeneration of axons that innervate the regrowing tail.