Tissue-engineered nerve guides with mesenchymal stem cells in the facial nerve regeneration.

Nerve guides with mesenchymal stem cells have been investigated in the rat facial nerve defect model to promote peripheral nerve regeneration and shorten recovery time to improve patients' quality of life. A 7-mm facial nerve gap experimental rat model is frequently employed in facial nerve regeneration studies. Facial nerve regeneration with nerve guides is evaluated by (1) assessing myelinated fiber counts using toluidine blue staining, (2) immunohistological analysis, (3) determining the g-ratio (axon diameter/total outer diameter) of regenerated nerve on transmission electron microscopic images, (4) retrograde nerve tracing in the facial nucleus, (5) electrophysiological evaluations using compound muscle action potential, and (6) functional evaluations using rat facial palsy scores. Dental pulp and adipose-derived stem cells, easily harvested using a minimally invasive procedure, possess characteristics of mesenchymal tissue lineages and can differentiate into Schwann-like cells. Cultured dental pulp-derived cells can produce neurotrophic factors, including nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. These neurotrophic factors promote peripheral nerve regeneration and afford protection against facial motor neuron death. Moreover, artificial nerve guides can maneuver axonal regrowth, and dental pulp-derived cells and adipose-derived Schwann cells may supply neurotrophic factors, promoting axonal regeneration. In the present review, the authors discuss facial nerve regeneration using nerve guides with mesenchymal stem cells.

Collagen/nano-sized ß-tricalcium phosphate conduits combined with collagen filaments and nerve growth factor promote facial nerve regeneration in miniature swine: an in vivo study.

OBJECTIVE: The aim of this study was to investigate the efficiency of a novel biomedical system that repairs facial nerve gaps in a miniature swine model. STUDY DESIGN: A collagen (COL)/nano-sized ß-tricalcium phosphate (nß-TCP) conduit combined with COL filaments and nerve growth factor (NGF) was prepared and used to bridge a 35-mm-long facial nerve gap in miniature swine. The functional recovery and axonal regeneration were evaluated by electrophysiologic and histologic assessments in the different groups at 6 months postoperatively. RESULTS: Morphologic analysis revealed that the COL+NGF, COL/nß-TCP+NGF, and autograft groups exhibited a superior recovery compared with the COL and COL/nß-TCP groups. The compound muscle action potential ratios were significantly greater in the COL/nß-TCP+NGF group compared to the COL+NGF and COL/nß-TCP groups (P < .01). Moreover, transmission electron microscopy demonstrated significantly larger axon diameters and myelin sheath thicknesses in the COL/nß-TCP+NGF group compared with the COL, COL+NGF, and COL/nß-TCP groups (P < .05). The expression of S-100 was significantly greater in the COL/nß-TCP+NGF group than in the COL+NGF and COL/nß-TCP groups (P < .05). CONCLUSIONS: The functional nerve biomedical system containing the COL/nß-TCP conduit combined with COL filaments and NGF could promote facial nerve regeneration, thus offering promising potential for clinical applications.

Effect of Motor versus Sensory Nerve Autografts on Regeneration and Functional Outcomes of Rat Facial Nerve Reconstruction.

Cranial nerve injury is disabling for patients, and facial nerve injury is particularly debilitating due to combined functional impairment and disfigurement. The most widely accepted approaches for reconstructing nerve gap injuries involve using sensory nerve grafts to bridge the nerve defect. Prior work on preferential motor reinnervation suggests, however, that motor pathways may preferentially support motoneuron regeneration after nerve injury. The effect of motor versus sensory nerve grafting after facial nerve injury has not been previously investigated. Insights into outcomes of motor versus sensory grafting may improve understanding and clinical treatment of facial nerve paralysis, mitigating facial asymmetry, aberrant reinnervation, and synkinesis. This study examined motor versus sensory grafting of the facial nerve to investigate effect of pathway on regeneration across a 5-mm rodent facial nerve defect. We enrolled 18 rats in 3 cohorts (motor, sensory, and defect) and recorded outcome measures including fiber count/nerve density, muscle endplate reinnervation, compound muscle action potential, and functional whisker twitch analysis. Outcomes were similar for motor versus sensory groups, suggesting similar ability of sensory and motor grafts to support regeneration in a clinically relevant model of facial nerve injury.

Morphometric properties of the facial nerve in fetal temporal bones.

OBJECTIVES: The main aim of the study was to examine the development and course of the facial nerve within fetal temporal bones from an anatomical and neuro-otological perspective. METHODS: The study was conducted on 32 temporal bones from obtained fetuses (7 females, 9 male), on a mean gestational age of 26.75 ±â€¯4.36 (range, 20-34) weeks from the collection of the Anatomy Department of Medicine Faculty. All the measurements were collected with a digital image analysis software. RESULTS: Neither male/female nor right/left significant differences were observed in relation with the algebraic data of the segment lengths and angles of the facial nerve (p > 0.05). Linear functions for meatal, labyrinthine, tympanic, and mastoid segment lengths of the facial nerve were calculated as: y = -1.206 + 0.200 × Age (weeks), y = -1.868 + 0.153 × Age (weeks), y = -2.327 + 0.325 × Age (weeks), and y = -1.507 + 0.246 × Age (weeks), respectively. In addition, linear functions for first and second genu angles were calculated as: y = 105.475-0.117 × Age (weeks) and y = 140.446-0.042 × Age (weeks), respectively. CONCLUSION: The regression equations and the scatter plot with increment curve, representing the growth dynamics of the facial nerve can be used for estimating its lengths and for understanding its development. The data suggest that there is a dramatic change transition from fetal life to the gathered data of adulthood in the length of meatal and mastoid segments as well as in the second genu angle; in addition, there is a partial change in the length of labyrinthine and tympanic segments as well as in the first genu angle.

The multiple sclerosis drug fingolimod (FTY720) stimulates neuronal gene expression, axonal growth and regeneration.

Fingolimod (FTY720) is a new generation oral treatment for multiple sclerosis (MS). So far, FTY720 was mainly considered to target trafficking of immune cells but not brain cells such as neurons. Herein, we analyzed FTY720's potential to directly alter neuronal function. In CNS neurons, we identified a FTY720 governed gene expression response. FTY720 upregulated immediate early genes (IEGs) encoding for neuronal activity associated transcription factors such as c-Fos, FosB, Egr1 and Egr2 and induced actin cytoskeleton associated genes (actin isoforms, tropomyosin, calponin). Stimulation of primary neurons with FTY720 enhanced neurite growth and altered growth cone morphology. In accordance, FTY720 enhanced axon regeneration in mice upon facial nerve axotomy. We identified components of a FTY720 engaged signaling cascade including S1P receptors, G12/13G-proteins, RhoA-GTPases and the transcription factors SRF/MRTF. In summary, we uncovered a broader cellular and therapeutic operation mode of FTY720, suggesting beneficial FTY720 effects also on CNS neurons during MS therapy and for treatment of other neurodegenerative diseases requiring neuroprotective and neurorestorative processes.

Double-powered free gracilis muscle transfer for smile reanimation: A longitudinal optoelectronic study.

The choice of the motor donor nerve is a crucial point in free flap transfer algorithms. In the case of unilateral facial paralysis, the contralateral healthy facial nerve can provide coordinated smile animation and spontaneous emotional expression, but with unpredictable axonal ingrowth into the recipient muscle. Otherwise, the masseteric nerve ipsilateral to the paralysis can provide a powerful neural input, without a spontaneous trigger of the smile. Harvesting a bulky muscular free flap may enhance the quantity of contraction but esthetic results are unpleasant. Therefore, the logical solution for obtaining high amplitude of smiling combined with spontaneity of movement is to couple the neural input: the contralateral facial nerve plus the ipsilateral masseteric nerve. Thirteen patients with unilateral dense facial paralysis underwent a one-stage facial reanimation with a gracilis flap powered by a double donor neural input, provided by both the ipsilateral masseteric nerve (coaptation by an end-to-end neurorrhaphy with the obturator nerve) and the contralateral facial nerve (coaptation through a cross-face nerve graft: end-to-end neurorrhaphy on the healthy side and end-to-side neurorrhaphy on the obturator nerve, distal to the masseteric/obturator neurorrhaphy). Their facial movements were evaluated with an optoelectronic motion analyzer. Before surgery, on average, the paretic side exhibited a smaller total three-dimensional mobility than the healthy side, with a 52% activation ratio and >30% of asymmetry. After surgery, the differences significantly decreased (analysis of variance (ANOVA), p < 0.05), with an activation ratio between 75% (maximum smile) and 91% (maximum smile with teeth clenching), and <20% of asymmetry. Similar modifications were seen for the performance of spontaneous smiles. The significant presurgical asymmetry of labial movements reduced after surgery. The use of a double donor neural input permitted both movements that were similar in force to that of the healthy side, and spontaneous movements elicited by emotional triggering.

Anatomic relationship between the facial nerve and the tympanic annulus.

OBJECTIVE: To identify the relationship between the vertical portion of the facial nerve and the tympanic annulus, using computed tomographic (CT) scans of healthy adult and pediatric patients. STUDY DESIGN: A retrospective review of CT scans. SETTING: The study was conducted in a tertiary referral medical center. PATIENTS: After excluding ears with noted pathology, a total of 241 ears were included in the final review (121 right and 120 left ears). The mean age of the patients was 33.3 years (2 mo to 87 yr). INTERVENTION(S): Both structures were identified at three distinct locations: the superior and the inferior most margins of the tympanic ring and at the umbo. Measurements were made in both anteroposterior and a mediolateral planes. Comparisons between different age groups were made to analyze the relative change in position that happens with age. RESULTS: The vertical portion of the facial nerve, as it proceeds distally, takes a more anterior and lateral course, and crosses the plane of the annulus almost consistently in the inferior third. The average distance at each location is reported. Statistically significant differences were found between age groups, in the areas around the second genu and the distal most segment of the facial nerve. CONCLUSIONS: This is the largest anatomic study to date analyzing the relationship of the facial nerve to the tympanic annulus. It also allows comparison between age groups, demonstrating a significant difference between adult and pediatric groups, especially in the distal portion of the fallopian canal.

Plasticity of mesenchymal stem cells from mouse bone marrow in the presence of conditioned medium of the facial nerve and fibroblast growth factor-2.

A number of evidences show the influence of the growth of injured nerve fibers in peripheral nervous system as well as potential implant stem cells (SCs). The SCs implementation in the clinical field is promising and the understanding of proliferation and differentiation is essential. This study aimed to evaluate the plasticity of mesenchymal SCs from bone marrow of mice in the presence of culture medium conditioned with facial nerve explants and fibroblast growth factor-2 (FGF-2). The growth and morphology were assessed for over 72 hours. Quantitative phenotypic analysis was taken from the immunocytochemistry for glial fibrillary acidic protein (GFAP), protein OX-42 (OX-42), protein associated with microtubule MAP-2 (MAP-2), protein ß-tubulin III (ß-tubulin III), neuronal nuclear protein (NeuN), and neurofilament 200 (NF-200). Cells cultured with conditioned medium alone or combined with FGF-2 showed morphological features apparently similar at certain times to neurons and glia and a significant proliferative activity in groups 2 and 4. Cells cultivated only with conditioned medium acquired a glial phenotype. Cells cultured with FGF-2 and conditioned medium expressed GFAP, OX-42, MAP-2, ß-tubulin III, NeuN, and NF-200. This study improves our understanding of the plasticity of mesenchymal cells and allows the search for better techniques with SCs.

Progressive postnatal motoneuron loss in mice lacking GDF-15.

Growth/differentiation factor-15 (GDF-15) is a widely expressed distant member of the TGF-beta superfamily with prominent neurotrophic effects on midbrain dopaminergic neurons. We show here that GDF-15-deficient mice exhibit progressive postnatal losses of spinal, facial, and trigeminal motoneurons. This deficit reaches a approximately 20% maximum at 6 months and is accompanied by losses of motor axons and significant impairment of rotarod skills. Similarly, sensory neurons in dorsal root ganglia (L4, L5) are reduced by 20%, whereas sympathetic neurons are not affected. GDF-15 is expressed and secreted by Schwann cells, retrogradely transported along adult sciatic nerve axons, and promotes survival of axotomized facial neurons as well as cultured motor, sensory, and sympathetic neurons. Despite striking similarities in the GDF-15 and CNTF knock-out phenotypes, expression levels of CNTF and other neurotrophic factors in the sciatic nerve were unaltered suggesting that GDF-15 is a genuine novel trophic factor for motor and sensory neurons.

Differential sensitivity of skeletal and fusimotor neurons to Bcl-2-mediated apoptosis during neuromuscular development.

Proper development of the nervous system requires that a carefully controlled balance be maintained between both proliferation and neuronal survival. The process of programmed cell death is believed to play a key role in regulating levels of neuronal survival, in large part through the action of antiapoptotic proteins, such as Bcl-2. Consistent with this, Bcl-2 has been shown to be a key regulator of apoptotic signaling in post-mitotic neurons. However, we still know remarkably little regarding the role that Bcl-2 plays in regulating the survival of specific motor neuron populations. In the present study, we have examined somatic motor neurons of the lumbar spinal cord, and branchiomotor neurons of the facial nucleus in bcl-2-null mice to determine the differential dependence among motor neuron populations with respect to Bcl-2-mediated survival. Examination of neuronal and axon number, axonal area, and the distribution of axonal loss in bcl-2-null mice demonstrates that, in contrast to the great majority of alpha motor neurons, gamma motor neurons exhibit a unique dependence upon bcl-2 for survival. These results demonstrate, for the first time, the connection between Bcl-2 expression, motor neuron survival, and the establishment of different motor populations.

Expression of ABCA2 protein in both non-myelin-forming and myelin-forming Schwann cells in the rodent peripheral nerve.

We previously reported that ABCA2, of the A subclass of the ATP-binding cassette (ABC) transporter superfamily, is expressed in mature oligodendrocytes and Schwann cells, the cells responsible for myelination in the brain and the peripheral nerve, respectively. However, unidentified cells expressing ABCA2 also were found. Here, we provide evidence for the expression of ABCA2 in the rodent sciatic nerve not only in Schwann cells, which express the Schwann cell marker S100beta and a zinc finger transcription factor Krox20 (a marker for myelin-forming Schwann cells), but also in Krox20-negative cells, which express glial fibrillary acidic protein (GFAP), a cell adhesion molecule L1, and S100beta weekly. We also analyzed developmental changes in ABCA2 expression in Schwann cells. The expression of ABCA2 in Krox20+/S100beta+ Schwann cells was found initially in rat facial nerve at postnatal day (PD) 8, in half (52.4%) of the cells showing myelinization at PD 14, and in all of the cells in the adult stage. These results demonstrate that ABCA2 is expressed in non-myelin-forming as well as in myelin-forming Schwann cells, and that ABCA2 may be involved in transport of a substance associated with cellular maturation rather than initial myelin formation in both types of Schwann cells.

Postnatal switching of NMDA receptor subunits from NR2B to NR2A in rat facial motor neurons.

The subunit composition of N-methyl-D-aspartate (NMDA) receptors affects their function under normal and pathological conditions. Functional NMDA receptors are expressed in lower motor neurons, but their subunit composition has not been defined. Here, we employed electrophysiology, quantitative PCR, and immunohistochemistry to investigate the subunit composition of NMDA receptors in postnatal motor neurons of the Wistar rat facial nucleus (FN). Whole-cell patch clamp recordings of acutely dissociated motor neurons from postnatal days 3 and 4 (P3-P4) showed that ifenprodil, a specific antagonist of the NMDA receptor 2B (NR2B) subunit, inhibited 91.62%+/-2.02% of NMDA-induced current, whereas NVP-AAM007, a specific antagonist of the NMDA receptor 2A (NR2A) subunit, inhibited much less of the current (16.69%+/-3.28%). Starting from P5, the inhibitory effects of ifenprodil and NVP-AAM007 gradually decreased and increased, respectively, such that the effect of NVP-AAM007 exceeded that of ifenprodil by P10. At P14, most of the NMDA-induced current was inhibited by NVP-AAM007 (84.59%+/-3.35%). Consistent with this, NR2B mRNA and protein were expressed highly at P3 and then gradually decreased by more than 75% by P14 in FN motor neurons, while NR1 was expressed stably over the same ages. However, NR2A mRNA and protein showed relatively constant levels between P3-P10 and decreased to 45% and 75% of the P3 level, respectively, by P14. Thus, analysis of functional NMDA receptors is critical to revealing subunit switching, which may be an important step in postnatal development of FN motor neurons.

Gustatory terminal field organization and developmental plasticity in the nucleus of the solitary tract revealed through triple-fluorescence labeling.

Early dietary sodium restriction has profound influences on the organization of the gustatory brainstem. However, the anatomical relationships among multiple gustatory nerve inputs have not been examined. Through the use of triple-fluorescence labeling and confocal laser microscopy, terminal fields of the greater superficial petrosal (GSP), chorda tympani (CT), and glossopharyngeal (IX) nerves were visualized concurrently in the nucleus of the solitary tract (NTS) of developmentally sodium-restricted and control rats. Dietary sodium restriction during pre- and postnatal development resulted in a twofold increase in the volume of both the CT and the IX nerve terminal fields but did not affect the volume of the GSP terminal field. In controls, these nerve terminal fields overlapped considerably. The dietary manipulation significantly increased the overlapping zones among terminal fields, resulting in an extension of CT and IX fields past their normal boundaries. The differences in terminal field volumes were exaggerated when expressed relative to the respective NTS volumes. Furthermore, increased terminal field volumes could not be attributed to an increase in the number of afferents because ganglion cell counts did not differ between groups. Taken together, selective increases in terminal field volume and ensuing overlap among terminal fields suggest an increased convergence of these gustatory nerve terminals onto neurons in the NTS. The genesis of such convergence is likely related to disruption of cellular and molecular mechanisms during the development of individual terminal fields, the consequences of which have implications for corresponding functional and behavioral alterations.

Synectin in the nervous system: expression pattern and potential as a binding partner of neurotrophin receptors.

To assess the potential for functional interaction between synectin and neurotrophin receptors (Trk receptors) in the nervous system, we characterized synectin expression in the rat brain. Synectin is widely expressed in the brain and its expression levels are regulated both temporally and spatially, correlating with those of Trk receptors. Biochemical studies indicated that synectin interacts with TrkB but not with TrkC in the developing brain. We also found that axotomized motoneurons upregulate synectin mRNA expression as well as TrkB mRNA. These data suggest that synectin plays a role in neural development and regeneration in association with TrkB.

The AP-1 transcription factor c-Jun is required for efficient axonal regeneration.

Nerve injury triggers numerous changes in the injured neurons and surrounding nonneuronal cells that ultimately result in successful target reinnervation or cell death. c-Jun is a component of the heterodimeric AP-1 transcription factor, and c-Jun is highly expressed in response to neuronal trauma. Here we have investigated the role of c-jun during axonal regeneration using mice lacking c-jun in the central nervous system. After transection of the facial nerve, the absence of c-Jun caused severe defects in several aspects of the axonal response, including perineuronal sprouting, lymphocyte recruitment, and microglial activation. c-Jun-deficient motorneurons were atrophic, resistant to axotomy-induced cell death, and showed reduced target muscle reinnervation. Expression of CD44, galanin, and alpha7beta1 integrin, molecules known to be involved in regeneration, was greatly impaired, suggesting a mechanism for c-Jun-mediated axonal growth. Taken together, our results identify c-Jun as an important regulator of axonal regeneration in the injured central nervous system.

Changes in mRNA of Slit-Robo GTPase-activating protein 2 following facial nerve transection.

Complex processes following peripheral nerve injury integrate a number of various external cues and their intracellular responses resulting in the cytoskeletal remodeling. One of these cues, Slit protein, plays an important role in neuronal migration and axonal guidance through the interaction with Roundabout (Robo) receptor. It was reported that the signal from Robo is transmitted to a specific family of GTPase-activating proteins (GAPs) named Slit-Robo GAPs. The Slit-Robo GAPs (srGAPs) further transmit the signal to the actin cytoskeleton controlling Rho GTPases and thus provide a direct link between Slit-Robo signaling and actin cytoskeleton. We examined the effects of facial nerve transection on srGAP2 mRNA expression in the facial nerve nuclei by in situ hybridization. SrGAP2 mRNA was initially expressed, and its expression increased from 3 to 28 days after transection, with the peak at the seventh day after axotomy. The upregulation was found mostly in the neuronal cells and only to a small extent in the glial cells. Our results suggest that srGAP2, as a part of Slit-Robo pathway, plays an important role in the axonal regeneration after axotomy.

Ontogeny of central CO2 chemoreception: chemosensitivity in the ventral medulla of developing bullfrogs.

Sites of central CO2 chemosensitivity were investigated in isolated brain stems from Rana catesbeiana tadpoles and frogs. Respiratory neurograms were made from cranial nerve (CN) 7 and spinal nerve 2. Superfusion of the brain stem with hypercapnic artificial cerebrospinal fluid elicited increased fictive lung ventilation. The effect of focal perfusion of hypercapnic artificial cerebrospinal fluid on discrete areas of the ventral medulla was assessed. Sites of chemosensitivity, which are active continuously throughout development, were identified adjacent to CN 5 and CN 10 on the ventral surface of the medulla. In early- and middle-stage tadpoles and frogs, unilateral stimulation within either site was sufficient to elicit the hypercapnic response, but simultaneous stimulation within both sites was required in late-stage tadpoles. The chemosensitive sites were individually disrupted by unilateral application of 1 mg/ml protease, and the sensitivity to bath application or focal perfusion of hypercapnia was reassessed. Protease lesions at CN 10 abolished the entire hypercapnic response, but lesions at CN 5 affected only the hypercapnic response originating from the CN 5 site. Neurons within the chemosensitive sites were also destroyed by unilateral application of 1 mM kainic acid, and the sensitivity to bath or focal application of hypercapnia was reassessed. Kainic acid lesions within either site abolished the hypercapnic response. Using a vital dye, we determined that kainic acid destroyed neurons by only within 100 microm of the ventral medullary surface. Thus, regardless of developmental stage, neurons necessary for CO2 sensitivity are located in the ventral medulla adjacent to CN 5 and 10.

In vivo application of mitochondrial pore inhibitors blocks the induction of apoptosis in axotomized neonatal facial motoneurons.

Axotomy induces apoptosis in motoneurons of neonatal rodents. To identify the key players in motoneuron apoptosis, we assessed the progression of apoptosis at 4 h intervals following facial motoneuron axotomy. The mitochondrial release of cytochrome c, caspase-3 activation and nuclear condensation were first observed in the motoneuron cell bodies 16 h postaxotomy. In vivo application of inhibitors of the mitochondrial permeability transition pore, Bongkrekic acid and cyclosporin A prevented cytochrome c release as well as caspase-3 activation and attenuated motoneuron apoptosis. Similarly, in vivo application of RU360, an inhibitor of the mitochondrial calcium uniporter, also protected axotomized motoneurons from apoptosis. Taken together, our results show that cytochrome c release and subsequent caspase-3 activation are critical events that precipitate the apoptotic death of axotomized neonatal motoneurons in vivo. In addition, these results provide evidence that application of mitochondrial pore inhibitors in vivo can block the induction of apoptosis following motoneuron axotomy.

Receptor protein tyrosine phosphatase sigma inhibits axonal regeneration and the rate of axon extension.

Transgenic mice lacking receptor protein tyrosine phophatase-sigma (RPTPsigma), a type IIa receptor protein tyrosine phosphatase, exhibit severe neural developmental deficits. Continued expression of RPTPsigma in the adult suggests that it plays a functional role in the mature nervous system. To determine if RPTPsigma might influence axonal regeneration, the time course of regeneration following facial nerve crush in wild-type and RPTPsigma (-/-) mice was compared. Mice lacking RPTPsigma exhibited an accelerated rate of functional recovery. Immunocytochemical examination of wild-type neurons in cell culture showed RPTPsigma protein in the growth cone. To determine if RPTPsigma affects the ability of a neuron to extend an axon, the rate of axon growth in neuronal cultures derived from wild-type and RPTPsigma (-/-) embryonic mice was compared. RPTPsigma did not affect the rate of axon initiation, but the rate of axon extension is enhanced in neurons obtained from RPTPsigma (-/-) mice. These findings indicate that RPTPsigma slows axon growth via a mechanism intrinsic to the neuron and identify a role for RPTPsigma regulating axonal regeneration by motoneurons.

Morphological study on the rootlets comprising the root of the intermediate nerve.

The number and arising sites of the rootlet comprising the intermediate nerve root were investigated in 100 sides of human brains. The arising sites of the rootlets were the pons halfway between the facial and vestibulocochlear nerves (31.9%), the arising portion of the vestibulocochlear nerve on the pons (28.8%) and the stem of the vestibulocochlear nerve (15.9%). The number of roots varied from one to five, with the most common being one root (58%). The number of rootlets per root also varied from one to five. Most of the roots had one rootlet (63%), while 2% of the roots had five rootlets. The variation of the arising sites of the intermediate nerve rootlets and its clinical significance were also discussed.