Persistence of tracer in the application site -a potential confounding factor in nerve regeneration studies

Selective reinnervation of peripheral targets after nerve injury might be assessed by injecting a first tracer in a target before nerve injury to label the original neuronal population, and applying a second tracer after the regeneration period to label the regenerated population. However, altered uptake of tracer, fading, and cell death may interfere with the results. Furthermore, if the first tracer injected remains in the target tissue, available for 're-uptake' by misdirected regenerating axons, which originally innervated another region, then the identification of the original population would be confused. With the aim of studying this problem, the sciatic nerve of adult rats was sectioned and sutured. After 3 days, to allow the distal axon to degenerate avoiding immediate retrograde transport, one of the dyes: Fast Blue (FB), Fluoro-Gold (FG) or Diamidino Yellow (DY), was injected into the tibial branch of the sciatic nerve, or in the skin of one of the denervated digits. Rats survived 2-3 months. The results showed labelled dorsal root ganglion (DRG) cells and motoneurones, indicating that late re-uptake of a first tracer occurs. This phenomenon must be considered when the model of sequential labelling is used for studying the accuracy of peripheral reinnervation

Efficacy of the fluorescent dyes Fast Blue, Fluoro-Gold, and Diamidino Yellow for retrograde tracing to dorsal root ganglia after subcutaneous injection

The present study was designed to investigate the efficacy of the fluorescent dyes Fast Blue (FB), Fluoro-Gold (FG), and Diamidino Yellow (DY) for retrograde tracing of lumbar dorsal root ganglia after their subcutaneous injection into different hindlimb digits. Injection of equal volumes (0.5 mu l) of 5% FB or 2% FG resulted in similar mean numbers of sensory neurones labelled by each tracer. Injection of equal volumes (0.5 mu l) of FB or FG in a single digit followed 10 days later by a second injection of the same volume of 5% DY into the same digit resulted in similar mean numbers of labelled sensory neurones for each of the three tracers. Furthermore, on average, 75% of all the FB-labelled cells and 74% of all FC-labelled cells also contained DY. Repeating the same experiment with an increased volume of DY (1.5 mu l) resulted in an increase in the mean number of double-labelled profiles to 82 and 84% for FB and FG, respectively. The results show that FB, FG and DY label similar numbers of cutaneous afferents and that a high level of double labelling may be obtained after sequential injections in digits. These properties make them suitable candidates in investigations where a combination of tracers with similar labelling efficacies is needed

Fast Blue and Diamidino Yellow as retrograde tracers in peripheral nerves: efficacy of combined nerve injection and capsule application to transected nerves in the adult rat

Capsule application of Diamidino Yellow (DY) to the cut end of the sciatic nerve immediately followed by capsule application of Fast Blue (FB) resulted in approximate to 95% double-labelled dorsal root ganglion neurones (DRGn) and motoneurones (Mn). Nerve injection of DY followed either immediately or 2 months later by capsule application of FB resulted in approximate to 90% double-labelled DRGn and Mn, indicating that DY and FB label similar populations of DRGn and Mn, and that insignificant DY fading occurred during this period. Inversing the order of application, however, i.e. nerve injection of FB followed immediately by capsule application of DY, resulted in double labelling in only approximate to 10% of the DRGn and Mn. These percentages increased to 70% of the DRGn and 60% of the Mn when the FB injection was followed 1 or 2 months after by the DY application, indicating that DY uptake is blocked by recent administration of FB. The results indicate that DY and FB might be useful for sequential labelling before and after nerve injury as a tool to investigate the accuracy of sensory and motor regeneration

Sciatic and femoral nerve sensory neurones occupy different regions of the L4 dorsal root ganglion in the adult rat.

The topographical distribution of sciatic and femoral nerve sensory neuronal somata in the L4 dorsal root ganglion of the adult rat was mapped after retrograde tracing with one or two of the dyes Fast Blue, Fluoro-Gold, or Diamidino Yellow. The tracers were applied to the proximal transected end of either nerve alone, or from both nerves in the same animal using separate tracers. Three-dimensional reconstructions of the distribution of labelled neurones were made from serial sections of the L4 dorsal root ganglion which is the only ganglion that these two nerves share. The results showed that with little overlap, femoral nerve neurones distribute dorsally and rostrally whereas sciatic nerve neurones distribute medially and ventrally. This finding indicates the existence of a somatotopical organisation for the representation of different peripheral nerves in dorsal root ganglia of adult animals

Heart rate variability analysis for the identification of the preictal interval in patients with drug-resistant epilepsy

Electrocardiogram (ECG) recordings, lasting hours before epileptic seizures, have been studied in the search for evidence of the existence of a preictal interval that follows a normal ECG trace and precedes the seizure's clinical manifestation. The preictal interval has not yet been clinically parametrized. Furthermore, the duration of this interval varies for seizures both among patients and from the same patient. In this study, we performed a heart rate variability (HRV) analysis to investigate the discriminative power of the features of HRV in the identification of the preictal interval. HRV information extracted from the linear time and frequency domains as well as from nonlinear dynamics were analysed. We inspected data from 238 temporal lobe seizures recorded from 41 patients with drug-resistant epilepsy from the EPILEPSIAE database. Unsupervised methods were applied to the HRV feature dataset, thus leading to a new perspective in preictal interval characterization. Distinguishable preictal behaviour was exhibited by 41% of the seizures and 90% of the patients. Half of the preictal intervals were identified in the 40 min before seizure onset. The results demonstrate the potential of applying clustering methods to HRV features to deepen the current understanding of the preictal state.FCT: CISUC -UID/CEC/00326/2020/ SFRH/BD/147862/2019info:eu-repo/semantics/publishedVersio

Representations of hindlimb digits in rat dorsal root ganglia

The distribution in dorsal root ganglia of neurones that innervate the distal tips of the hindlimb digits in the rat were mapped after subcutaneous injections of the fluorescent tracers Fast Blue, Diamidino Yellow, and Fluoro-Gold into different digits. Three-dimensional reconstruction was used to describe the intraganglionic distribution of neurones labelled from different digits. Labelled neurones were found mainly in the L3-L5 ganglia. The distribution in ganglia and the number of neurones labelled from each digit varied considerably between cases, but mean numbers of labelled neurones were similar for the different digits. Neurones in L3 tended to innervate medial digits and neurones in L5 tended to innervate lateral digits, but most neurones from any digit were found in L4. Although overlap was considerable, the three-dimensional reconstruction showed tendencies of neurones to be distributed in restricted territories within the dorsal root ganglia. This was especially clear in ganglion L5, where digit TV was found to be represented more rostrally than digit V. The results indicate that primary afferent neurones that innervate the hindlimb digits are represented by a crude rostrocaudal somatotopic organisation both among and within lumbar dorsal root gangli

Differential methylation of TCF7L2 promoter in peripheral blood DNA in newly diagnosed, drug-naïve patients with type 2 diabetes

TCF7L2 is the susceptibility gene for Type 2 diabetes (T2D) with the largest effect on disease risk that has been discovered to date. However, the mechanisms by which TCF7L2 contributes to the disease remain largely elusive. In addition, epigenetic mechanisms, such as changes in DNA methylation patterns, might have a role in the pathophysiology of T2D. This study aimed to investigate the differences in terms of DNA methylation profile of TCF7L2 promoter gene between type 2 diabetic patients and age- and Body Mass Index (BMI)- matched controls. We included 93 type 2 diabetic patients that were recently diagnosed for T2D and exclusively on diet (without any pharmacological treatment). DNA was extracted from whole blood and DNA methylation was assessed using the Sequenom EpiTYPER system. Type 2 diabetic patients were more insulin resistant than their matched controls (mean HOMA IR 2.6 vs 1.8 in controls, P<0.001) and had a poorer beta-cell function (mean HOMA B 75.7 vs. 113.6 in controls, P<0.001). Results showed that 59% of the CpGs analyzed in TCF7L2 promoter had significant differences between type 2 diabetic patients and matched controls. In addition, fasting glucose, HOMA-B, HOMA-IR, total cholesterol and LDL-cholesterol correlated with methylation in specific CpG sites of TCF7L2 promoter. After adjustment by age, BMI, gender, physical inactivity, waist circumference, smoking status and diabetes status uniquely fasting glucose, total cholesterol and LDL-cholesterol remained significant. Taken together, newly diagnosed, drug-naïve type 2 diabetic patients display specific epigenetic changes at the TCF7L2 promoter as compared to age- and BMI-matched controls. Methylation in TCF7L2 promoter is further correlated with fasting glucose in peripheral blood DNA, which sheds new light on the role of epigenetic regulation of TCF7L2 in T2D

Gastric inhibitory polypeptide receptor methylation in newly diagnosed, drug-naïve patients with type 2 diabetes: a case-control study

GIP action in type 2 diabetic (T2D) patients is altered. We hypothesized that methylation changes could be present in GIP receptor of T2D patients. This study aimed to assess the differences in DNA methylation profile of GIPR promoter between T2D patients and age- and Body Mass Index (BMI)-matched controls. We included 93 T2D patients (cases) that were uniquely on diet (without any anti-diabetic pharmacological treatment). We matched one control (with oral glucose tolerance test negative, non diabetic), by age and BMI, for every case. Cytokines and hormones were determined by ELISA. DNA was extracted from whole blood and DNA methylation was assessed using the Sequenom EpiTYPER system. Our results showed that T2D patients were more insulin resistant and had a poorer β cell function than their controls. Fasting adiponectin was lower in T2D patients as compared to controls (7.0±3.8 µgr/mL vs. 10.0±4.2 µgr/mL). Levels of IL 12 in serum were almost double in T2D patients (52.8±58.3 pg/mL vs. 29.7±37.4 pg/mL). We found that GIPR promoter was hypomethylated in T2D patients as compared to controls. In addition, HOMA-IR and fasting glucose correlated negatively with mean methylation of GIPR promoter, especially in T2D patients. This case-control study confirms that newly diagnosed, drug-naïve T2D patients are more insulin resistant and have worse β cell function than age- and BMI-matched controls, which is partly related to changes in the insulin-sensitizing metabolites (adiponectin), in the proinflammatory profile (IL12) and we suggest in the methylation pattern of GIPR. Our study provides novel findings on GIPR promoter methylation profile which may improve our ability to understand type 2 diabetes pathogenesis

Sentinel-1 DInSAR for Monitoring Active Landslides in Critical Infrastructures: The Case of the Rules Reservoir (Southern Spain)

We thank the editors and four anonymous reviewers for helpful comments and suggestions that improved the manuscript.Landslides in reservoir contexts are a well-recognised hazard that may lead to dangerous situations regarding infrastructures and people’s safety. Satellite-based radar interferometry is proving to be a reliable method to monitor the activity of landslides in such contexts. Here, we present a DInSAR (Differential Interferometric Synthetic Aperture Radar) analysis of Sentinel-1 images that exemplifies the usefulness of the technique to recognize and monitor landslides in the Rules Reservoir (Southern Spain). The integration of DInSAR results with a comprehensive geomorphological study allowed us to understand the typology, evolution and triggering factors of three active landslides: Lorenzo-1, Rules Viaduct and El Arrecife. We could distinguish between rotational and translational landslides and, thus, we evaluated the potential hazards related to these typologies, i.e., retrogression (Lorenzo-1 and Rules Viaduct landslides) or catastrophic slope failure (El Arrecife Landslide), respectively. We also observed how changes in the water level of the reservoir influence the landslide’s behaviour. Additionally, we were able to monitor the stability of the Rules Dam as well as detect the deformation of a highway viaduct that crosses a branch of the reservoir. Overall, we consider that other techniques must be applied to continue monitoring the movements, especially in the El Arrecife Landslide, in order to avoid future structural damages and fatalities.A Spanish “Sistema de Garantía Juvenil” research contract, founded by the Junta de Andalucía and the European Social Funds, supported the work of Cristina Reyes-Carmona. Spanish “Ramón y Cajal” grant supported part of the work of Jorge Pedro Galve. This work has been partially funded by the Spanish Ministry of Economy and Competitiveness through the DEMOS project “Deformation monitoring using Sentinel-1 data” (Ref: CGL2017-83704-P) and the LITHOSURF project “Respuesta de la topografía y la red de drenaje a procesos litosféricos y climáticos en el sur de Iberia” (Ref: CGL2015-67130-C2-1-R). This work has been partially developed in the framework of the RISKCOAST project (Ref: SOE3/P4/E0868) funded by the Interreg SUDOE program (3rd call for proposals)