Cerebrospinal fluid volume and nerve root vulnerability during lumbar puncture or spinal anaesthesia at different vertebral levels.

Cerebrospinal fluid (CSF) and nerve root volumes within the lumbosacral dural sac were estimated at various vertebral levels, in an attempt to determine any possible relevance to the incidence of nerve root trauma during lumbar puncture or spinal anaesthesia. Magnetic resonance images from seven patients were studied. Volumes were calculated by semi-automatic threshold segmentation combined with manual editing of each slice. The mean dural sac volume from S1 to T12 was 42.8±5.8 ml and the mean CSF volume 34.3±5.1 ml with the mean root volume being 10.4±2.2 cm(3). The mean CSF volume per vertebral segment ranged from 4.3±0.7 ml at L5, to 5.8±2.5 ml at L1, with high inter-individual variability. The mean root volume ranged from 0.6±0.1 cm(3) at L5 to 2.4±0.5 cm(3) at T12. The conus medullaris was located at L1 in four of the five patients scanned at upper lumbar levels, and at the lower border of L2 in the other. Vulnerability to nerve root damage was expressed as the Vulnerability Index (%), being defined as the ratio of root volume to dural sac volume (CSF volume + root volume). The value ranged between 7 and 14% at L5, increasing rostrally to 30 to 43% at T12. Caution is obviously required in high punctures to avoid contact with the conus medullaris, but the cauda equina is also vulnerable to contact with more caudal punctures and had a Vulnerability Index of about 25% at L4, that increased rostrally.

Three-dimensional magnetic resonance image of structures enclosed in the spinal canal relevant to anesthetists and estimation of the lumbosacral CSF volume.

Three-dimensional (3D) image-reconstruction of structures inside the spinal canal certainly produces relevant data of interest in regional anesthesia. Nowadays, all hospital MRI equipment is designed mainly for clinical diagnostic purposes. In order to overcome the limitations we have produced more accurate images of structures contained inside the spinal canal using different software, validating our quantitative results with those obtained with standard hospital MRI equipment. Neuroanatomical 3D reconstruction using Amira software, including detailed manual edition was compared with semi-automatic 3D segmentation for CSF volume calculations by commonly available software linked to the MR equipment (MR hospital). Axial sections from seven patients were grouped in two aligned blocks (T1 Fast Field Eco 3D and T2 Balance Fast Field Eco 3D-resolution 0,65 x 0,65 x 0,65 mm, 130 mm length, 400 sections per case). T2 weighted was used for CSF volume estimations. The selected program allowed us to reconstruct 3D images of human vertebrae, dural sac, epidural fat, CSF and nerve roots. The CSF volume, including the amount contained inside nerve roots, was calculated. Different segmentation thresholds were used, but the CSF volume estimations showed high correlation between both teams (Pearson coefficient = 0.98, p = 0.003 for lower blocks; Pearson 0.89, p = 0.042 for upper blocks). The mean estimated value of CSF volume in lower blocks (L3-S1) was 15.8 + 2.9 ml (Amira software) and 13.1 +/- 1.9 ml (software linked to the MR equipment) and in upper blocks (T11-L2) was 21 +/- 4.47 ml and 18.9 +/- 3.5 ml, respectively. A high variability was detected among cases, without correlation with either weight, height or body mass index. Aspects concerning the partial volume effect are also discussed. Quick semi-automatic hospital 3D reconstructions give results close to detailed neuroanatomical 3D reconstruction and could be used in the future for individual quantification of lumbosacral CSF volumes and other structures for anesthetic purposes.

[Structure of the arachnoid layer of the human spinal meninges: a barrier that regulates dural sac permeability]. / Morfología de la lámina aracnoidea espinal humana. Barrera que limita la permeabilidad del saco dural.

OBJECTIVES: Drugs injected into the epidural space are known to penetrate the subarachnoid space by simple diffusion through the dural sac. We aimed to study the cellular ultrastructure of the arachnoid membrane and the type of intercellular junctions responsible for creating the barrier that regulates the passage of drugs through the dural sac in humans. MATERIAL AND METHODS: Fourteen tissue samples of arachnoid membrane were taken from 2 patients during procedures that required opening the lumbar dural sac. The samples were treated with glutaraldehyde, osmium tetroxide, ferrocyanide and acetone, and then embedded in resin. Ultrathin sections were stained with lead citrate for examination by transmission electron microscopy. RESULTS: The arachnoid membrane was 35 to 40 microm thick. The outer surface contained neurothelial cells (dural border cells) along the subdural compartment, while the internal portion was made up of a plane 5 to 8 microm thick with 4 to 5 arachnoid cells overlapping to form a barrier layer. The intercellular spaces on this plane were 0.02 to 0.03 microm wide; the arachnoid cells were bridged by specialized junctions (desmosomes and other tight junctions). CONCLUSIONS: Structural features of the arachnoid cells provide a barrier within the human dural sac. They occupy only the internal portion of the arachnoid membrane. Specialized intercellular junctions explain the selective permeability of this membrane.

Morfología de la lámina aracnoidea espinal humana. Barrera que limita la permeabilidad del saco dural / Structure of the arachnoid layer of the human spinal meninges: a barrier that regulates dural sac permeability

OBJETIVOS: Se ha demostrado que las moléculas inyectadas en el espacio epidural pasan desde éste al espacio subaracnoideo por difusión simple a través de la pared del saco dural. Nuestro objetivo fue estudiar la ultraestructura de células de la lámina aracnoidea y tipo de uniones especializadas responsables del efecto barrera que gobierna el tránsito de moléculas a través del saco dural humano. MATERIAL Y MÉTODO: Se estudiaron catorce muestras de la lámina aracnoidea obtenidas de dos pacientes durante intervenciones con apertura del saco dural lumbar. Las muestras se trataron con glutaraldehido, tetróxido de osmio, ferrocianuro, acetona, e incluyeron en resina. Los cortes ultrafinos se contrastaron con citrato de plomo, para poder ser observados con un microscopio electrónico de transmisión. RESULTADOS: La lámina aracnoidea posee un espesor de 35-40 μm. En su porción externa se hallan células neuroteliales del compartimento subdural, mientras que su porción interna está formada por un plano celular de 5-8 μm de espesor, constituido por la superposición de 4-5 células aracnoideas que forman la capa barrera. El espacio intercelular de este plano fue de 0,02-0,03 μm. Entre las células aracnoideas se encontraron uniones especializadas de membrana de tipo desmosomas y uniones estrechas. CONCLUSIONES: Las células aracnoideas poseen características estructurales que aseguran la función barrera del saco dural humano y no ocupan todo el espesor de la lámina aracnoidea, sólo su porción interna. La presencia de uniones especializadas de membrana entre sus células justifica la permeabilidad selectiva de esta lámina(AU)

OBJETIVES: Drugs injected into the epidural space are known to penetrate the subarachnoid space by simple diffusion through the dural sac. We aimed to study the cellular ultrastructure of the arachnoid membrane and the type of intercellular junctions responsible for creating the barrier that regulates the passage of drugs through the dural sac in humans. MATERIAL AND METHODS: Fourteen tissue samples of arachnoid membrane were taken from 2 patients during procedures that required opening the lumbar dural sac. The samples were treated with glutaraldehyde, osmium tetroxide, ferrocyanide and acetone, and then embedded in resin. Ultrathin sections were stained with lead citrate for examination by transmission electron microscopy. RESULTS: The arachnoid membrane was 35 to 40 μm thick. The outer surface contained neurothelial cells (dural border cells) along the subdural compartment, while the internal portion was made up of a plane 5 to 8 μm thick with 4 to 5 arachnoid cells overlapping to form a barrier layer. The intercellular spaces on this plane were 0.02 to 0.03 μm wide; the arachnoid cells were bridged by specialized junctions (desmosomes and other tight junctions). CONCLUSIONS: Structural features of the arachnoid cells provide a barrier within the human dural sac. They occupy only the internal portion of the arachnoid membrane. Specialized intercellular junctions explain the selective permeability of this membrane(AU)

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.

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.

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.

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.

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.