Anna Puigdellívol-Sánchez

On the use of fast blue, fluoro-gold and diamidino yellow for retrograde tracing after peripheral nerve injury: uptake, fading, dye interactions, and toxicity.

The usefulness of three retrograde fluorescent dyes for tracing injured peripheral axons was investigated. The rat sciatic was transected bilaterally and the proximal end briefly exposed to either Fast Blue (FB), Fluoro-Gold (FG) or to Diamidino Yellow (DY) on the right side, and to saline on the left side, respectively. The nerves were then resutured and allowed to regenerate. Electrophysiological tests 3 months later showed similar latencies and amplitudes of evoked muscle and nerve action potentials between tracer groups. The nerves were then cut distal to the original injury and exposed to a second (different) dye. Five days later, retrogradely labelled neurones were counted in the dorsal root ganglia (DRGs) and spinal cord ventral horn. The number of neurones labelled by the first tracer was similar for all three dyes in the DRG and ventral horn except for FG, which labelled fewer motoneurones. When used as second tracer, DY labelled fewer neurones than FG and FB in some experimental situations. The total number of neurones labelled by the first and/or second tracer was reduced by about 30% compared with controls. The contributions of cell death as well as different optional tracer combinations for studies of nerve regeneration are discussed.

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.

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 IV 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 ganglia. J. Comp. Neurol. 408:137–145, 1999.

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 approximately 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 approximately 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 approximately 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.

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. Injections of equal volumes (0.5 microl) of 51% FB or 2% FG resulted in similar mean numbers of sensory neurones labelled by each tracer. Injection of equal volumes (0.5 microl) 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 FG-labelled cells also contained DY. Repeating the same experiment with an increased volume of DY (1.5 microl) 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.

Contribution of femoral and proximal sciatic nerve branches to the sensory innervation of hindlimb digits in the rat

The present study was performed to investigate the possibility of “aberrant” innervation of the tips of the hindlimb digits in the rat, i.e., from other sources than the femoral and the main sciatic branches (tibial, peroneal, sural). Cutaneous injections of fluorescent tracers in the digits were combined with either selective nerve transections to restrict afferent routes followed by detection of labeled neurons in dorsal root ganglia (DRGs), or by a delayed application of a second tracer to afferent nerves under study to detect double labeled neurons in DRGs. The results show that the tips of the digits were represented in DRGs L3–6. The femoral nerve afferents from digits 1 and 2 projected primarily to DRG L3 and to a smaller extent to DRG L4. A small number of neurons from primarily medial digits 1 and 2, but also from lateral digits 3‐5, were found to project to DRGs L4 and L5 via a proximal branch that leaves the sciatic nerve near the sciatic notch and runs distally in the posterior part of the thigh, here called the musculocutaneous nerve of the hindlimb. We also have some evidence indicating innervation of the tips of the digits from the posterior cutaneous nerve of the thigh. Aberrant innervation such as that described here might contribute to remaining and perhaps abnormal sensibility after nerve injury and is of interest for the interpretation of results in experimental studies of collateral and regenerative sprouting after such injury. Anat Rec 260:180–188, 2000.

3D interactive model of lumbar spinal structures of anesthetic interest

A 3D model of lumbar structures of anesthetic interest was reconstructed from human magnetic resonance (MR) images and embedded in a Portable Document Format (PDF) file, which can be opened by freely available software and used offline. The MR images were analyzed using a specific 3D software platform for biomedical data. Models generated from manually delimited volumes of interest and selected MR images were exported to Virtual Reality Modeling Language format and were presented in a PDF document containing JavaScript‐based functions. The 3D file and the corresponding instructions and license files can be downloaded freely at http://diposit.ub.edu/dspace/handle/2445/44844?locale=en. The 3D PDF interactive file includes reconstructions of the L3–L5 vertebrae, intervertebral disks, ligaments, epidural and foraminal fat, dural sac and nerve root cuffs, sensory and motor nerve roots of the cauda equina, and anesthetic approaches (epidural medial, spinal paramedial, and selective nerve root paths); it also includes a predefined sequential educational presentation. Zoom, 360° rotation, selective visualization, and transparency graduation of each structure and clipping functions are available. Familiarization requires no specialized informatics knowledge. The ease with which the document can be used could make it valuable for anatomical and anesthetic teaching and demonstration of patient information. Clin. Anat. 28:205–212, 2015.

Morfología de la lámina aracnoidea espinal humana. Barrera que limita la permeabilidad del saco dural

Resumen Objetivos Se ha demostrado que las moleculas inyectadas en el espacio epidural pasan desde este al espacio subaracnoideo por difusion simple a traves de la pared del saco dural. Nuestro objetivo fue estudiar la ultraestructura de celulas de la lamina aracnoidea y tipo de uniones especializadas responsables del efecto barrera que gobierna el transito de moleculas a traves del saco dural humano Material y metodo Se estudiaron catorce muestras de la lamina aracnoidea obtenidas de dos pacientes durante intervenciones con apertura del saco dural lumbar. Las muestras se trataron con glutaraldehido, tetroxido de osmio, ferrocianuro, acetona, e incluyeron en resina. Los cortes ultrafinos se contrastaron con citrato de plomo, para poder ser observados con un microscopio electronico de transmision Resultados La lamina aracnoidea posee un espesor de 35-40 μm. En su porcion externa se hallan celulas neuroteliales del compartimento subdural, mientras que su porcion interna esta formada por un plano celular de 5-8 μm de espesor, constituido por la superposicion de 4-5 celulas aracnoideas que forman la capa barrera. El espacio inter-celular de este plano fue de 0,02-0,03 μm. Entre las celulas aracnoideas se encontraron uniones especializadas de membrana de tipo desmosomas y uniones estrechas Conclusiones Las celulas aracnoideas poseen caracteristicas estructurales que aseguran la funcion barrera del saco dural humano y no ocupan todo el espesor de la lamina aracnoidea, solo su porcion interna. La presencia de uniones especializadas de membrana entre sus celulas justifica la permeabilidad selectiva de esta lamina

Unintentional subdural placement of epidural catheters during attempted epidural anesthesia: an anatomic study of spinal subdural compartment.

Background: Although infrequent, subdural block is a complication of epidural anesthesia with obvious implications. Knowledge of the spinal subdural compartment (dura-arachnoid interface) may help elucidate controversies arising from evidence that subdural catheter placement is feasible and may be difficult to identify clinically. Methods: Samples of arachnoid lamina obtained during in vivo lumbosacral surgery (n = 4) and from cadavers (n = 6) were obtained and prepared for transmission electron microscopy and scanning electron microscopy. Subdural spaces were artificially produced in suitable samples, and an epidural catheter was inserted between the arachnoid and dura to compare the dimensions of meninges in relation to epidural catheters. Results: Scanning electron microscopy of the dural sac showed areas of continuity between the arachnoid lamina and dura mater and other parts with both membranes separated by a subdural space. Transmission electron microscopy allowed the study of such border zones, where alternating cellular and collagen layers could be seen. A layer rich in collagen fibers and some fibroblasts separated arachnoid and neurothelial cells (dural border cells). Few specialized membrane junctions were found among cells adjacent to collagen fibers. Dura mater had an average thickness of 260 to 400 &mgr;m, with a dural lamina of approximately 4 to 6 &mgr;m. In areas where the arachnoid appeared separated from the dural lamina, its thickness measured 35 to 45 &mgr;m. Catheters with a diameter of 700 &mgr;m were successfully inserted inside the subdural space, between the dura mater and the arachnoid lamina. Conclusions: Dura mater and arachnoid layers act as a single unit but may be pulled apart by traction forces during cadaver processing of the dural sac or in vivo placement of catheters. This generates subdural spaces, either parallel or concentric, because of the minimal resistance offered by the tissue, which may be explained by its few specialized membrane junctions.

Estimations of topographically correct regeneration to nerve branches and skin after peripheral nerve injury and repair

Peripheral nerve injury is typically associated with long-term disturbances in sensory localization, despite nerve repair and regeneration. Here, we investigate the extent of correct reinnervation by back-labeling neuronal soma with fluorescent tracers applied in the target area before and after sciatic nerve injury and repair in the rat. The subpopulations of sensory or motor neurons that had regenerated their axons to either the tibial branch or the skin of the third hindlimb digit were calculated from the number of cell bodies labeled by the first and/or second tracer. Compared to the normal control side, 81% of the sensory and 66% of the motor tibial nerve cells regenerated their axons back to this nerve, while 22% of the afferent cells from the third digit reinnervated this digit. Corresponding percentages based on quantification of the surviving population on the experimental side showed 91%, 87%, and 56%, respectively. The results show that nerve injury followed by nerve repair by epineurial suture results in a high but variable amount of topographically correct regeneration, and that proportionally more neurons regenerate into the correct proximal nerve branch than into the correct innervation territory in the skin.