Neurotrophins reduce degeneration of injured ascending sensory and corticospinal motor axons in adult rat spinal cord

Spinal cord regeneration in adult mammals is limited by neurite outgrowth inhibitors and insufficient availability of outgrowth-promoting agents. Formation of degenerative swellings at the proximal ends of severed axons (terminal clubs), which starts early after injury, also may hinder recovery and their rupture may contribute to secondary spinal cord damage. We investigated whether neurotrophins would reduce these degenerative processes. Adult rats received a transection of the dorsal column sensory and corticospinal motor tracts at T9 and anterograde tracing of the axons from the sciatic nerve and motor cortex, respectively. The highest number of terminal clubs was found at 1 day and approximately half remained present until at least 28 days. A single injection immediately after injury of a mixture of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 into the lesion site, reduced the number of terminal clubs in the sensory system by approximately half at 1 and 7 days (but not 14) after the lesion. Individual or combinations of two neurotrophins were as effective, suggesting that the neurotrophins protected similar axonal populations. The injected neurotrophins did not affect degeneration of corticospinal motor axons. A 7-day continuous intrathecal infusion of neurotrophin-3 was more effective and also reduced terminal club formation of corticospinal axons by approximately 60%. Spinal tissue loss was not affected by the neurotrophin treatments, suggesting that terminal clubs are not major contributors to the pathogenesis of secondary spinal degeneration during the first two weeks. Thus, neurotrophins can reduce axonal degeneration in the spinal cord after traumatic axonal injury.     

The importance of transgene and cell type on the regeneration of adult retinal ganglion cell axons within reconstituted bridging grafts

When grafted onto the cut optic nerve, chimeric peripheral nerve (PN) sheaths reconstituted with adult Schwann cells (SCs) support the regeneration of adult rat retinal ganglion cell (RGC) axons. Regrowth can be further enhanced by using PN containing SCs transduced ex vivo with lentiviral (LV) vectors encoding a secretable form of ciliary neurotrophic factor (CNTF). To determine whether other neurotrophic factors or different cell types also enhance RGC regrowth in this bridging model, we tested the effectiveness of (1) adult SCs transduced with brain-derived neurotrophic factor (BDNF) or glial cell line-derived neurotrophic factor (GDNF), and (2) fibroblasts (FBs) genetically modified to express CNTF. SCs transduced with LV-BDNF and LV-GDNF secreted measurable and bioactive amounts of each of these proteins, but reconstituted grafts containing LV-BDNF or LV-GDNF transduced SCs did not enhance RGC survival or axonal regrowth. LV-BDNF modified grafts did, however, contain many pan-neurofilament immunolabeled axons, many of which were also immunoreactive for calcitonin gene-related peptide (CGRP) and were presumably of peripheral sensory origin. Nor-adrenergic and cholinergic axons were also seen in these grafts. There were far fewer axons in LV-GDNF engineered grafts. Reconstituted PN sheaths containing FBs that had been modified to express CNTF did not promote RGC viability or regeneration, and PN reconstituted with a mixed population of SCs and CNTF expressing FBs were less effective than SCs alone. These data show that both the type of neurotrophic factor and the cell types that express these factors are crucial elements when designing bridging substrates to promote long-distance regeneration in the injured CNS.     

Increased brain-derived neurotrophic factor immunoreactivity in rat dorsal root ganglia and spinal cord following peripheral inflammation

Our recent study showed that peripheral inflammation induced an increased expression of brain-derived neurotrophic factor (BDNF) mRNA which was mediated by nerve growth factor (NGF) in the dorsal root ganglion (DRG). In the present study, we evaluated the change of BDNF immunoreactivity in the DRG and spinal cord following peripheral inflammation by means of immunohistochemistry. Significant increases in the percentage of BDNF-immunoreactive (IR) neuron profiles in the L5 DRG and marked elevation in the expression of BDNF-IR terminals in the spinal dorsal horn were observed following peripheral tissue inflammation produced by an intraplantar injection of Freund's adjuvant into the rat paws. These findings suggest that peripheral tissue inflammation induces an increased BDNF synthesis in the DRG and an elevated anterograde transport of BDNF to the spinal dorsal horn. The functional role of this increased BDNF was discussed briefly.     

Potential regulation by trophic factors of low-affinity NGF receptors in spinal motor neurons

Developing spinal motor neurons (SMN) express low-affinity nerve growth factor receptors (LNGFR) but not high-affinity transducing NGF receptors. Moreover, SMN are not supported by NGF in vitro. In the normal adult rat most SMN are not LNGFR immunoreactive (LNGFR-IR), but they transiently reexpress LNGFR (though not the high-affinity receptor) after peripheral nerve injury. With a cut lesion of the sciatic nerve (when only a neuroma forms), the number of LNGFR-IR SMN at L4–L6 rapidly increases to a maximum between day 1 and 7 and returns to baseline levels by day 30. After a crush lesion (accompanied by regeneration to the muscle), LNGFR-IR SMN appear in about the same numbers, but they start to disappear 1 week later. We speculate that the similar appearance and differential decline of LNGFR-IR seen after the two types of lesions are regulated by the availability of a common signal such as ciliary neurotrophic factor. The adult SMN model provides a good opportunity to investigate the reexpression of LNGFR after peripheral nerve injury, and more generally, the unknown role and regulation of LNGFR.     

NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord

The regeneration capacity of spinal cord axons is severely limited. Recently, much attention has focused on promoting regeneration of descending spinal cord pathways, but little is known about the regenerative capacity of ascending axons. Here we have assessed the ability of neurotrophic factors to promote regeneration of sensory neurons whose central axons ascend in the dorsal columns. The dorsal columns of adult rats were crushed and either brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) or a vehicle solution was delivered continuously to the lesion site for 4 weeks. Transganglionic labelling with cholera toxin beta subunit (CTB) was used to selectively label large myelinated Abeta fibres. In lesioned rats treated with vehicle, CTB-labelled fibres were observed ascending in the gracile fasciculus, but these stopped abruptly at the lesion site, with no evidence of sprouting or growth into lesioned tissue. No CTB-labelled terminals were observed in the gracile nucleus, indicating that the lesion successfully severed all ascending dorsal column axons. Treatment with BDNF did not promote axonal regeneration. In GDNF-treated rats fibres grew around cavities in caudal degenerated tissue but did not approach the lesion epicentre. NT-3, in contrast, had a striking effect on promoting growth of lesioned dorsal column axons with an abundance of fibre sprouting apparent at the lesion site, and many fibres extending into and beyond the lesion epicentre. Quantification of fibre growth confirmed that only in NT-3-treated rats did fibres grow into the crush site and beyond. No evidence of terminal staining in the gracile nucleus was apparent following any treatment. Thus, although NT-3 promotes extensive growth of lesioned axons, other factors may be required for complete regeneration of these long ascending projections back to the dorsal column nuclei. The intrathecal delivery of NT-3 or other neurotrophic molecules has obvious advantages in clinical applications, as we show for the first time that dorsal column axonal regeneration can be achieved without the use of graft implantation or nerve lesions.     

Patients with temporal lobe epilepsy show an increase in brain-derived neurotrophic factor protein and its correlation with neuropeptide Y

Model studies on animal seizures have proposed potential involvement of the neurotrophins, BDNF and NGF, in human epilepsy. However, their biological significance in this disease itself remains to be evaluated. Here we demonstrate that patients with intractable temporal lobe epilepsy show a marked increase in protein levels of BDNF (2.6-fold, p<0.01) but not other neurotrophins. Moreover, the specific BDNF increase was significantly correlated with contents of neuropeptide Y. Thus, these results indicate the activity-dependent expression of BDNF in human subjects and its potential contribution to the pathophysiology of human epilepsy via neuropeptide Y.     

Release of immunoreactive brain-derived neurotrophic factor in the spinal cord of the rat following sciatic nerve transection

Using the antibody microprobe method, the sites of spinal release of immunoreactive brain-derived neurotrophic factor (BDNF) was studied in normal rats, and rats with prior sciatic nerve transection. In normal rats, a significant basal release of immunoreactive BDNF was found in the superficial dorsal horn. Following sciatic nerve transection (performed 14 days previously), release of BDNF was found throughout the whole of the dorsal horn, extending into deeper laminae. Electrical stimulation of the ipsilateral sciatic nerve at a strength adequate to excite either A fibres (20 Hz at 2x threshold voltage) or A and C fibres (2 Hz at 20x threshold voltage) did not alter the basal release of immunoreactive BDNF in normal or in nerve-injured rats. The results suggest that BDNF is released from the central terminals of primary afferent fibres, but such release is not solely dependent upon action potential invasion of these terminals. The increased extent of release following nerve transection is consistent with the hypothesis that BDNF plays a role in the central response to peripheral nerve injury.     

Persistence of anti-NGF induced dorsal root axons: possible penetration into the mammalian spinal cord

Neonatal rats were given daily injections of antisera to nerve growth factor protein (anti-NGF) for a period of 1 month and then allowed to survive 17 more months. The number of neurons in dorsal root ganglia (DRG) and axons in the dorsal root (DR) were determined in the anti-NGF rats and compared to similar numbers from untreated littermates. We found a 32% decrease in DRG neuron number and 32 and 34% increases in myelinated and unmyelinated DR fibers, respectively, in the anti-NGF rats. The sensory cell bodies in the anti-NGF rats were on the average 23% larger than in the normal rats. We conclude that in an NGF deprived environment a population of DRG neurons dies, principally the small neurons, and in response the surviving neurons emit extra processes which persist for most of the life of the rat. This suggests that the anti-NGF induced axons enter the spinal cord and synapse.     

Brain-derived neurotrophic factor (BDNF) prevents lesion-induced axonal die-back in young rat optic nerve

Lesions of the optic nerve in young animals lead to rapid retrograde degeneration of the axon stumps and to death of retinal ganglion cells. We injected different neurotrophic factors into the eye at the time of an intracranial freeze-crush lesion of the optic nerve in 8 day old rats. Optic nerve axons were visualized by anterograde tracing with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and by electron microscopy. The lesion induced a rapid die-back of the axons, which could be prevented by BDNF and to a lesser extent by neurotrophin-3 (NT-3) or ciliary neurotrophic factor (CNTF). No effect was seen in animals injected with nerve growth factor (NGF) or a mixture of acidic and basic fibroblast growth factor (FGF). In contrast to this effect on the axons, none of these factors was able to counteract the rapidly progressing degeneration of the retinal ganglion cells. These results suggest a selective influence of BDNF on the mechanisms responsible for the maintenance of optic nerve axons.     

The impact of motor and sensory nerve architecture on nerve regeneration

Sensory nerve autografting is the standard of care for injuries resulting in a nerve gap. Recent work demonstrates superior regeneration with motor nerve grafts. Improved regeneration with motor grafting may be a result of the nerve's Schwann cell basal lamina tube size. Motor nerves have larger SC basal lamina tubes, which may allow more nerve fibers to cross a nerve graft repair. Architecture may partially explain the suboptimal clinical results seen with sensory nerve grafting techniques. To define the role of nerve architecture, we evaluated regeneration through acellular motor and sensory nerve grafts. Thirty-six Lewis rats underwent tibial nerve repairs with 5 mm double-cable motor or triple-cable sensory nerve isografts. Grafts were harvested and acellularized in University of Wisconsin solution. Control animals received fresh motor or sensory cable isografts. Nerves were harvested after 4 weeks and histomorphometry was performed. In 6 animals per group from the fresh motor and sensory cable graft groups, weekly walking tracks and wet muscle mass ratios were performed at 7 weeks. Histomorphometry revealed more robust nerve regeneration in both acellular and cellular motor grafts. Sensory groups showed poor regeneration with significantly decreased percent nerve, fiber count, and density (p < 0.05). Walking tracks revealed a trend toward improved functional recovery in the motor group. Gastrocnemius wet muscle mass ratios show a significantly greater muscle mass recovery in the motor group (p < 0.05). Nerve architecture (size of SC basal lamina tubes) plays an important role in nerve regeneration in a mixed nerve gap model.     

Application of weak electric field to the hindpaw enhances sciatic motor nerve regeneration in the adult rat

Direct current (DC) electrical stimulation of the hindpaw is shown to enhance sciatic motor nerve regeneration in the adult rat. Cathodal stimulation, using weak currents (10 microA/cm2; field strength approximately 100 mV/cm) increased the reinnervation of the hindpaw muscles as measured by evoked electromyograms. This enhanced regeneration only occurred after cut-and-suture lesions, but not after crushing injury of the sciatic nerve. This enhancement of motor nerve regeneration by weak DC fields had been previously described in amphibians but we are the first to describe this phenomenon in mammals.     

Neuro-glial interactions in the adult rat retina after reaxotomy of ganglion cells: examination of neuron survival and phagocytic microglia using fluorescent tracers

Retinal ganglion cells (RGCs) regenerating through peripheral nerve grafts show enhanced survival after further axonal injury for at least 4 weeks [Restor. Neurol. Neurosci. 21 (2003) 11]. Here, we examined the survival of the neurons and their microglial phagocytosis in dependence of the site of reaxotomy. Therefore, the optic nerve in adult rats was transected at different distances from the eye cup and replaced with an autologous piece of sciatic nerve. After 14 days of axonal growth, the regenerated neurites were reaxotomized either within the remaining optic stump or within the graft and their cell bodies were retrogradely labeled. Reaxotomy of regenerated ganglion cells within the remaining optic nerve resulted in reduced (but not significant) ganglion cell survival and significant microglial phagocytosis in contrast to reaxotomy within the peripheral nerve graft. Furthermore, phagocytosis-dependent labeling using two different fluorescent tracers revealed that the same microglial cell can phagocytose further dying ganglion cells within 14 days after the first activation. The results suggest that the intrasciatic segments of axons receive some trophic support that is retrogradely transported and required to limit the microglial activation. The microglial capability to phagocytose dying neurons several fold emphasizes their function in permanent scavenging within the retina.     

Adenosine inhibition of the regeneration in vitro of adult frog sciatic sensory axons

The sensory axons of the adult frog sciatic nerve have earlier been shown to regenerate in vitro. If a local test crush is made at the initiation of culturing, regeneration starts after 3.4 days and proceeds at a rate of about 0.8–0.9 mm/day for several days. In the present experiments regeneration was inhibited by adenosine in a reversible and dose-dependent fashion. Similarly, both an adenosine analogue, 2-chloroadenosine (2-CA), and a non-hydrolyzable ATP analogue, AMP-PNP, reduced the outgrowth of sensory axons. The effect of adenosine was partially antagonized by theophylline at a critical concentration. Using a compartmental system, it could clearly be shown that adenosine exerted its effects at the outgrowth region. Adenosine, 2-CA, and AMP-PNP were also found to inhibit the proliferation of Schwann cells in the regenerating nerve. Various experiments showed that the latter can not explain the outgrowth inhibitory effects, which could be mediated by adenosine receptors associated with the elongating axons.     

Neurotrophins alter the numbers of neurotransmitter-ir mature vagal/glossopharyngeal visceral afferent neurons in vitro

Mature nodose and petrosal ganglia neurons (placodally derived afferent neurons of the vagal and glossopharyngeal nerves) contain TrkA and TrkC, and transport specific neurotrophins [nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4)]. This study evaluated neurotrophin influences on the presence of neuropeptides and/or neurotransmitter enzymes in these visceral sensory neurons. NGF, NT-3 and NT-4 (10–100 ng/ml) were applied (5 days) to dissociated, enriched, cultures of mature nodose/petrosal ganglia neurons, and the neurons processed for tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neurofilament (NF-200) immunocytochemistry. Addition of NGF to nodose/petrosal ganglia neuron-enriched cultures significantly increased the number of TH-immunoreactive (ir) neurons, decreased the number of VIP-ir neurons in the cultures, and did not affect the numbers of CGRP-ir neurons. The addition of an NGF neutralizing antibody attenuated the effects of NGF on TH and VIP-ir neurons. NT-3 increased the number of VIP-ir neurons in the nodose/petrosal ganglia cultures and did not alter the numbers of TH-, or CGRP-ir neurons. The addition of an NT-3 neutralizing antibody attenuated the effects of NT-3 on VIP-ir neurons. NT-4 had no significant effects on the numbers of TH, VIP and CGRP-ir neurons. The absence of neurotrophin-induced changes in the numbers of NF-200-ir neurons in culture showed the lack of neurotrophin-mediated changes in survival of mature vagal afferent neurons. These data demonstrate that specific neurotrophins influence the numbers of neurons labeled for specific neurochemicals in nodose/petrosal ganglia cultures. These data, coupled with previous evidence for the presence of TrkA and TrkC mRNA and of the retrograde transport of NGF and NT-3, suggest important roles for NGF and NT-3 in the maintenance of transmitter phenotype of these mature visceral afferent neurons.     

Neurotrophins from dorsal root ganglia trigger allodynia after spinal nerve injury in rats

Injury to peripheral nerves often results in chronic pain which is difficult to relieve. The mechanism underlying the pain syndrome remains largely unknown. In previous studies we showed that neurotrophins are up-regulated in satellite cells around sensory neurons following sciatic nerve lesion. In the present study, we have examined whether the neurotrophins in the dorsal root ganglia play any role in allodynia after nerve injury. Antibodies to different neurotrophins, directly delivered to injured dorsal root ganglia, significantly reduced (with different time sequences) the percentage of foot withdrawal responses evoked by von Frey hairs. The antibodies to nerve growth factor acted during the early phase but antibodies to neurotrophin-3 and brain-derived neurotrophic factor were effective during the later phase. Exogenous nerve growth factor or brain-derived neurotrophic factor, but not neurotrophin-3, directly delivered to intact dorsal root ganglia, trigger a persistent mechanical allodynia. Our results showed that neurotrophins within the dorsal root ganglia after peripheral nerve lesion are involved in the generation of allodynia at different stages. These studies provide the first evidence that ganglia-derived neurotrophins are a source of nociceptive stimuli for neuropathic pain after peripheral nerve injury.     

Specific and nonspecific regeneration of motor axons after sciatic nerve injury and repair in the rat

The pattern of motor axon regeneration following unilateral sciatic nerve lesions (freezing or transection) was studied in adult rats. Transected nerves were repaired with epineurial or fascicular sutures. Four months after the lesion, the motor neuron cell body localization in the spinal cord of plantar or common peroneal nerve axons were examined bilaterally with retrograde transport of horseradish peroxidase. Motor neuron cell body localization was similar bilaterally after freezing, indicating that regenerating axons had reached their original peripheral innervation territory. However, after nerve transection, irrespective of whether epineurial or fascicular sutures were used, motor neuron cell body distribution on the operated side was abnormal with numerous labeled cell bodies located outside the area of the normal motor neuron pool. This finding indicates that after nerve transection the normal pattern of motor axon innervation is not restored even after fascicular nerve repair.     

Contribution of degeneration of motor and sensory fibers to pain behavior and the changes in neurotrophic factors in rat dorsal root ganglion

To elucidate the role of the degeneration of motor and sensory fibers in neuropathic pain, we examined the pain-related behaviors and the changes of brain-derived neurotrophic factor (BDNF) in the L4/5 dorsal root ganglion (DRG) and the spinal cord after L5 ventral rhizotomy. L5 ventral rhizotomy, producing a selective lesion of motor fibers, produced thermal hyperalgesia and increased BDNF expression in tyrosine kinase A-containing small- and medium-sized neurons in the L5 DRG and their central terminations within the spinal cord, but not in the L4 DRG. Furthermore, L5 ventral rhizotomy up-regulated nerve growth factor (NGF) protein in small to medium diameter neurons in the L5 DRG and also in ED-1-positive cells in the L5 spinal nerve, suggesting that NGF synthesized in the degenerative fibers is transported to the L5 DRG and increases BDNF synthesis. On the other hand, L5 ganglionectomy, producing a selective lesion of sensory fibers, produced heat hypersensitivity and an increase in BDNF and NGF in the L4 DRG. These data indicate that degeneration of L5 sensory fibers distal to the DRG, but not motor fibers, might influence the neighboring L4 nerve fibers and induce neurotrophin changes in the L4 DRG. We suggest that these changes of neurotrophins in the intact primary afferents of neighboring nerves may be one of many complex mechanisms, which can explain the abnormal pain behaviors after nerve injury. The ventral rhizotomy and ganglionectomy models may be useful to investigate the pathophysiological mechanisms of neuropathic pain after Wallerian degeneration in motor or sensory or mixed nerve.     

The effect of brain-derived neurotrophic factor on sensory and autonomic function after lingual nerve repair

Brain-derived neurotrophic factor (BDNF) is important in the response to peripheral nerve injury and may enhance regeneration. We have assessed its role in the functional recovery of sensory afferents and autonomic efferents after repair of the chorda tympani and lingual nerves in the cat. Six months after entubulation repair, with or without the incorporation of BDNF at the repair site, the recovery of secretomotor and vasomotor efferents was determined by recording salivary flow from the submandibular gland and temperature changes on the tongue surface, each evoked by stimulation of the repaired nerve. Electrophysiological recordings from the lingual and chorda tympani nerves proximal to the repair were undertaken to characterise mechanosensitive, thermosensitive, and gustatory afferents. When compared with data from uninjured control animals, both repair groups showed changes in receptor sensitivity and spontaneous discharge, and persistent reductions in conduction velocity, proportion of gustatory and thermosensitive units, rate of salivary secretion, and vasomotor responses. Comparisons between the outcome of repair with or without BDNF revealed few differences. In the BDNF group, fewer units in the chorda tympani responded to gustatory or thermal stimuli and the sensitivity of the gustatory units was lower. The conduction velocity of afferents in the lingual nerve was also lower, but the mechanoreceptive field size was higher. Thus, despite its known trophic role in the gustatory system, BDNF had not enhanced recovery of these or other fibre populations. We conclude that the application of BDNF to a site of lingual nerve repair has a negative effect on the long-term outcome.     

大鼠局灶性脑缺血后神经营养因子表达的自然变化

目的 观察大鼠局灶性脑缺血后大鼠神经行为、梗死体积、组织形态及缺血半暗带神经生长因子(NGF)、脑源性神经营养因子(BDNF)的表达水平变化。方法 将健康雄性SD大鼠24只随机分为2组,Ⅰ组(假手术组); Ⅱ组(脑缺血组)。用线栓法建立动物模型,不给予再灌注,各组在术后48h断头取脑,处死前行神经功能评分,用氯化三苯基四氮唑(TTC)染色计算脑梗死体积,用HE染色观察组织学形态,用免疫组织化学染色观察NGF、BDNF的表达水平。结果 Ⅰ组在神经功能评分、梗死体积、组织形态及大脑皮层相应部位NGF、BDNF阳性细胞数均正常。与Ⅰ组比较,Ⅱ组的神经功能评分和梗死体积均严重受损; 光镜下Ⅱ组缺血性病理改变较重,与Ⅰ组比较,Ⅱ组缺血半暗带NGF、BDNF阳性神经元数增加(P<0.05)。结论 脑缺血本身可上调缺血半暗带NGF、BDNF的表达水平。     

Intrathecal administration of nerve growth factor delays GAP 43 expression and early phase regeneration of adult rat peripheral nerve

Whether nerve growth factor (NGF) promotes peripheral nerve regeneration in vivo, in particular in adults, is controversial. We therefore examined the effect of exogenous NGF on nerve regeneration and the expression of GAP 43 (growth-associated protein 43) in adult rats. NGF was infused intrathecally via an osmotic mini-pump, while control rats received artificial cerebrospinal fluid. Two days after the infusion was initiated, the right sciatic nerves were transected or crushed, and the animals allowed to survive for 3 to 11 days. The right DRG, the right proximal stump of the transected sciatic nerve, and the posterior horn of the spinal cord were examined by Western blotting, immunohistochemistry, and electron microscopy. GAP 43 immunoreactivity in the NGF-treated animals was significantly lower than in the aCSF-treated controls. Electron microscopy showed that the number of myelinated and unmyelinated axons decreased significantly in the NGF-treated rats as compared with the controls. These findings are indicative that exogenous NGF delayed GAP 43 induction and the early phase of peripheral nerve regeneration and supports the hypothesis that the loss of NGF supply from peripheral targets via retrograde transport caused by axotomy serves as a signal for DRG neurons to invoke regenerative responses. NGF administered intrathecally may delay the neurons' perception of the reduction of the endogenous NGF, causing a delay in conversion of DRG neurons from the normal physiological condition to regrowth state.