Claes Hildebrand

Molecular Signaling and Pulpal Nerve Development

The purpose of this review is to discuss molecular factors influencing nerve growth to teeth. The establishment of a sensory pulpal innervation occurs concurrently with tooth development. Epithelial/mesenchymal interactions initiate the tooth primordium and change it into a complex organ. The initial events seem to be controlled by the epithelium, and subsequently, the mesenchyme acquires odontogenic properties. As yet, no single initiating epithelial or mesenchymal factor has been identified. Axons reach the jaws before tooth formation and form terminals near odontogenic sites. In some species, local axons have an initiating function in odontogenesis, but it is not known if this is also the case with mammals. In diphyodont mammals, the primary dentition is replaced by a permanent dentition, which involves a profound remodeling of terminal pulpal axons. The molecular signals underlying this remodeling remain unknown. Due to the senescent deterioration of the dentition, the target area of tooth nerves shrinks with age, and these nerves show marked pathological-like changes. Nerve growth factor and possibly also brain-derived neurotrophic factor seem to be important in the formation of a sensory pulpal innervation. Neurotrophin-3 and -4/5 are probably not involved. In addition, glial cell line-derived neurotrophic factor, but not neurturin, seems to be involved in the control of pulpal axon growth. A variety of other growth factors may also influence developing tooth nerves. Many major extracellular matrix molecules, which can influence growing axons, are present in developing teeth. It is likely that these molecules influence the growing pulpal axons.

Changes in a rat facial muscle after facial nerve injury and repair.

This study describes changes in a rat facial muscle innervated by the mandibular and buccal facial nerve branches 4 months after nerve injury and repair. The following groups were studied: (A) normal controls; (B) spontaneous reinnervation by collateral or terminal sprouting; (C) reinnervation after surgical repair of the mandibular branch; and (D) chronic denervation. The normal muscle contained 1200 exclusively fast fibers, mainly myosin heavy chain (MyHC) IIB fibers. In group B, fiber number and fiber type proportions were normal. In group C, fiber number was subnormal. Diameters and proportions of MyHC IIA and hybrid fibers were above normal. The proportion of MyHC IIB fibers was subnormal. Immediate and delayed repair gave similar results with respect to the parameters examined. Group D rats underwent severe atrophic and degenerative changes. Hybrid fibers prevailed. These data suggest that spontaneous regeneration of the rat facial nerve is superior to regeneration after surgical repair and that immediacy does not give better results than moderate delay with respect to surgical repair. Long delays are shown to be detrimental.

The developmental skeletal growth in the rat foot is reduced after denervation

Abstract It has long been known that bone is innervated. In recent years it has been suggested that the local nerves may influence the growth and metabolism of bone by way of neuropeptides. The transient local presence of nerve-containing cartilage canals just before formation of secondary ossification centres in rat knee epiphyses seems to support that view. The purpose of the present study was to see if denervation affects the developmental growth of metatarsal bones in the rat hindfoot. We made sciatic and femoral neurectomies in 7- day-old rat pups and examined the hindfeet at various times after surgery. Immunohistochemical analysis showed that denervation was complete. Radiographic examination revealed that the metatarsal bones were significantly shorter in denervated hindfeet 30 days after denervation (average relative shortening 9.9±2.3%). Measurements of total foot length showed that denervated feet were subnormally sized already five days postoperatively, before the onset of secondary ossification. The timing of the latter was not affected by denervation. Control rats subjected to tenotomies exhibited normal metatarsal bone lengths. On the basis of these results we suggest that the local nerves may influence the growth of immature bones but do not affect secondary ossification.

Neuropathy in diabetic BB/Wor rats treated with insulin implants

Abstract To elucidate the pathophysiology of diabetic neuropathy many workers have examined nerve specimens from diabetic rats. While most workers found that animals with high blood glucose levels develop neuropathy, some researchers report that the peripheral nerves are normal in hyperglycaemic rats. Hypoglycaemia may also cause neuropathy. Some workers suggest that neuropathy is linked to fluctuations of the blood glucose level. In the present study we examine plantar nerves of diabetic BB/ Wor rats maintained on an eu-/hyperglycaemic or an eu-/ hypoglycaemic regime with insulin implants. Treatment with implants worked well. Light microscopic examination of nerve fibres in non-diabetic control rats and in eu-/ hyperglycaemic diabetic rats showed a normal picture. Preparations from eu-/hypoglycemic rats showed irregular myelin sheaths and signs of Wallerian degeneration. The lengths and diameters of the largest internodes were significantly subnormal. We conclude, that periodic moderate hypoglycaemia, but not periodic moderate hyperglycaemia, elicits neuropathy in diabetic BB/Wor rats treated with insulin implants.

Occurrence of epidermal nerve endings in glabrous and hairy skin of the rat foot after sciatic nerve regeneration

Abstract.The occurrence and distribution of intraepidermal nerve endings in hairy and glabrous skin of the rat foot was examined in normal cases and three months after sciatic neurotomy/suture or a crush lesion. The nerve endings were visualized in cryostate sections with antibodies against protein gene product 9.5. Normal glabrous skin exhibited 23.3 endings/mm length. Neurotomy/suture cases had 6.1 endings/mm. In rats with nerve crush injury the occurrence was normal, but the intraepidermal nerve endings tended to be abnormally short and occurred mainly in the basal layer of the epidermis. In sections from hairy skin countings were not possible. Subjective evaluation indicated that the occurrence of dermal and epidermal axon profiles usually was very deficient after neurotomy/suture and essentially normal after crush. Skin samples from the contralateral side of operated animals showed a normal occurrence and distribution of nerve endings. We also noted that cases subjected to neurotomy/suture showed increased numbers of protein gene product 9.5-immunoreactive intraepidermal cells and an abnormally thin epidermis. We suggest that a highly deficient occurrence of intraepidermal nerve endings may be one factor behind the unsatisfactory restitution of the sensory function of the hand typically seen in patients subjected to neurotomy and suture

Hypoglycaemic neuropathy in BB/Wor rats treated with insulin implants: electron microscopic observations.

Abstract Insulin-dependent diabetes mellitus is a chronic metabolic disease that causes long-term secondary complications such as neuropathy. The occurrence of diabetic neuropathy has generally been thought of as being associated with hyperglycaemia. However, in a previous light microscopic examination of plantar nerves in diabetic BB/Wor rats treated with insulin implants we found that eu-/hyperglycaemic rats present a normal picture, whereas eu-/hypoglycaemic rats show severe changes. The aim of the present work is to supplement our previous light microscopic report with electron microsocpic data from the lateral plantar nerve of normal, eu-/hyperglycaemic and eu-/hypoglycaemic BB/Wor rats. Under the electron microscope lateral plantar nerves collected from eu-/hyperglycaemic rats presented a qualitatively normal picture. In addition, the fibre numbers and the size distribution of the myelinated fibres were normal. In contrast, specimens from eu-/hypoglycaemic BB/Wor rats showed severe qualitative changes, interpreted as signs of axonal de- and regeneration. The total number of axons was somewhat subnormal and the sizes of the myelinated fibres were strongly shifted towards smaller diameters. These data confirm our previous light microscopic observations. We conclude that eu-/hypoglycaemic BB/Wor rats treated with insulin implants, but not similarly treated eu-/hyperglycaemic animals, develop a neuropathy in their plantar nerves.

Molecular heterogeneity of oligodendrocytes in chicken white matter

The classical studies by Del Rio Hortega (Mem. Real. Soc. Espan. Hist. Nat. 14:40–122, 1928) suggest that the oligodendrocyte population includes four morphological subtypes. Recent data from the cat and the rat show that the anatomy of oligodendrocytes related to early myelinating prospective large fibers differs from that of oligodendrocytes related to late myelinating prospective small fibers. After application of a polyclonal antiserum to cryostat sections from the chicken CNS, we noted that glial cells in the spinal cord white matter had become labeled. Analysis of the occurrence and cellular localization of this immunoreactivity—the T4‐O immunoreactivity—in the CNS of the adult chicken showed that T4‐O immunoreactive cells are enriched in the ventral funiculus and superficially in the lateral funiculus of the spinal cord, where they are co‐localized with large fibers. Double staining with T4‐O antiserum and anti‐GFAP or the lectin BSI‐B4 revealed that T4‐O immunoreactive cells are not astrocytes or microglia. Staining with anti‐HSP108, a general marker for avian oligodendrocytes, showed that T4‐O immunoreactivity defines an oligodendroglial subpopulation. A search for T4‐O immunoreactivity in spinal cord white matter of some other vertebrates revealed that T4‐O immunoreactive cells are not present in sections from fish, frog, turtle, rat, and rabbit spinal cord white matter. These results suggest the presence of a fiber size‐related molecular heterogeneity among chicken white matter oligodendrocytes. GLIA 27:15–21, 1999.

Myelination of prospective large fibres in chicken ventral funiculus

In mammals, the oligodendrocyte population includes morphological and molecular varieties. We reported previously that an antiserum against the T4-O molecule labels a subgroup of oligodendrocytes related to large myelinated axons in adult chicken white matter. We also reported that T4-O immunoreactive cells first appear in the developing ventral funiculus (VF) at embryonic day (E)15, subsequently increasing rapidly in number. Relevant fine structural data for comparison are not available in the literature. This prompted the present morphological analysis of developing and mature VF white matter in the chicken. The first axon-oligodendrocyte connections were seen at E10 and formation of compact myelin had started at E12. Between E12 and E15 the first myelinating oligodendrocytes attained a Schwann cell-like morphology. At hatching (E21) 60% of all VF axons were myelinated and in the adult this proportion had increased to 85%. The semilunar or polygonal oligodendrocytes associated with adult myelinated axons contained many organelles indicating a vivid metabolic activity. Domeshaped outbulgings with gap junction-like connections to astrocytic profiles were frequent. Oligodendrocytes surrounded by large myelinated axons and those surrounded by small myelinated axons were cytologically similar. But, thick and thin myelin sheaths had dissimilar periodicities and Marchi-positive myelinoid bodies occurred preferentially in relation to large myelinated axons. We conclude that early oligodendrocytes contact axons and form myelin well before the first expression of T4-O and that emergence of a T4-O immunoreactivity coincides in time with development of a Type IV phenotype. Our data also show that oligodendrocytes associated with thick axons are cytologically similar to cells related to thin axons. In addition, the development of chicken VF white matter was found to be similar to the development of mammalian white matter, except for the rapid time course.

Neuropeptide effects on rat chondrocytes and perichondrial cells in vitro.

This study examines if cultured chondrocytes and perichondrial cells change the level of cAMP and/or cGMP in response to application of the neuropeptide calcitonin gene-related peptide (CGRP). Cells collected from the knee region of 4-8 days old rat pups were cultured in vitro. Cultures were exposed to 10(-10)-10(-6) M CGRP during 10 minutes. The levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in the cultures and in controls were determined by radioimmunoassay. The results show that application of CGRP causes a distinctly increased level of cAMP, that was absent when CGRP was applied together with the CGRP(1) receptor antagonist. The level of cGMP was not obviously altered. Hence, it is possible that terminals of primary sensory neurones present in developing cartilage influence chondrocytes and perichondrial cells via local release of CGRP.

Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro

Our previous finding that skin-derived and muscle-derived molecules can be used to sort regenerating rat sciatic nerve axons evoked questions concerning neuron-target interactions at the level of single cells, which prompted the present study. The results show that dorsal root ganglion (DRG) neurons co-cultured with fibroblast-like skin-derived cells emit many neurites. These have a proximal linear segment and a distal network of beaded branches in direct relation to skin-derived cells. Electron microscopic examination of such co-cultures showed bundles of neurites at some distance from the target cells and single profiles closely apposed to subjacent cells. RNase protection assay revealed that cultivated skin-derived cells express nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In co-cultures of DRG neurons and 3T3 fibroblasts overexpressing either of the neurotrophins produced by skin-derived cells the picture varied. NT-3 transfected 3T3 fibroblasts gave a growth pattern similar to that seen with skin-derived cells. Neurons co-cultured with mock-transfected 3T3 fibroblasts were small and showed weak neurite growth. In co-cultures with a membrane insert between skin-derived cells or 3T3 fibroblasts and DRG neurons few neurons survived and neurite growth was very sparse. We conclude that skin-derived cells stimulate neurite growth from sensory neurons in vitro, that these cells produce NGF, BDNF, NT-3 and NT-4 and that 3T3 fibroblasts producing NT-3 mimic the effect of skin-derived cells on sensory neurons in co-culture. Finally the results suggest that cell surface molecules are important for neuritogenesis.