Corneal nerves: structure, contents and function.

The TFOS DEWS II Pathophysiology Subcommittee reviewed the mechanisms involved in the initiation and perpetuation of dry eye disease. Its central mechanism is evaporative water loss leading to hyperosmolar tissue damage. Research in human disease and in animal models has shown that this, either directly or by inducing inflammation, causes a loss of both epithelial and goblet cells. The consequent decrease in surface wettability leads to early tear film breakup and amplifies hyperosmolarity via a Vicious Circle. Pain in dry eye is caused by tear hyperosmolarity, loss of lubrication, inflammatory mediators and neurosensory factors, while visual symptoms arise from tear and ocular surface irregularity. Increased friction targets damage to the lids and ocular surface, resulting in characteristic punctate epithelial keratitis, superior limbic keratoconjunctivitis, filamentary keratitis, lid parallel conjunctival folds, and lid wiper epitheliopathy. Hybrid dry eye disease, with features of both aqueous deficiency and increased evaporation, is common and efforts should be made to determine the relative contribution of each form to the total picture. To this end, practical methods are needed to measure tear evaporation in the clinic, and similarly, methods are needed to measure osmolarity at the tissue level across the ocular surface, to better determine the severity of dry eye. Areas for future research include the role of genetic mechanisms in non-Sjogren syndrome dry eye, the targeting of the terminal duct in meibomian gland disease and the influence of gaze dynamics and the closed eye state on tear stability and ocular surface inflammation.

TFOS DEWS II pathophysiology report

C-fos is a proto-oncogene that is expressed within some neurons following depolarization. The protein product, c-fos protein, can be identified by immunohistochemical techniques. Therefore, c-fos expression might be used as a marker for neuronal activity throughout the neuraxis following peripheral stimulation. This study has analyzed patterns of c-fos expression in both control and anesthetized animals and in anesthetized rats subjected to various forms of peripheral stimulation. Labeled cells were counted in the spinal cord, brainstem, hypothalamus, and thalamus. Little c-fos immunoreactivity was found in control animals. Prolonged inhalational anesthesia increased the number of labeled cells at several brainstem sites. Noxious stimulation of anesthetized rats induced c-fos within the neuraxis in patterns consistent with data obtained from electrophysiological studies and in additional locations for which few direct electrophysiological data are available, such as the ventrolateral medulla, the posterior hypothalamic nucleus, and the reuniens and paraventricular thalamic nuclei. Gentle mechanical stimulation was ineffective in inducing c-fos-like protein. The data suggest that c-fos can be used as a transynaptic marker for neuronal activity following noxious stimulation. However, c-fos is expressed only in some kinds of neurons following peripheral stimulation, and it therefore may be an incomplete marker for nociresponsive activity. In addition, at least a few neurons express c-fos protein in the absence of noxious stimulation. Experiments analyzing c-fos expression must be designed with care, as both extraneous stimuli and anesthetic depth influence the results.

Expression of C-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat

Neural pathways underlying vocal control.

The peripheral sensory nerves that innervate the cornea can be easily damaged by trauma, surgery, infection or diabetes. Several growth factors and axon guidance molecules, such as Semaphorin3A (Sema3A) are upregulated upon cornea injury. Nerves can regenerate after injury but do not recover their original density and patterning. Sema3A is a well known axon guidance and growth cone repellent protein during development, however its role in adult cornea nerve regeneration remains undetermined. Here we investigated the neuro-regenerative potential of Sema3A on adult peripheral nervous system neurons such as those that innervate the cornea. First, we examined the gene expression profile of the Semaphorin class 3 family members and found that all are expressed in the cornea. However, upon cornea injury there is a fast increase in Sema3A expression. We then corroborated that Sema3A totally abolished the growth promoting effect of nerve growth factor (NGF) on embryonic neurons and observed signs of growth cone collapse and axonal retraction after 30 min of Sema3A addition. However, in adult isolated trigeminal ganglia or dorsal root ganglia neurons, Sema3A did not inhibited the NGF-induced neuronal growth. Furthermore, adult neurons treated with Sema3A alone produced similar neuronal growth to cells treated with NGF and the length of the neurites and branching was comparable between both treatments. These effects were replicated in vivo, where thy1-YFP neurofluorescent mice subjected to cornea epithelium debridement and receiving intrastromal pellet implantation containing Sema3A showed increased corneal nerve regeneration than those receiving pellets with vehicle. In adult PNS neurons, Sema3A is a potent inducer of neuronal growth in vitro and cornea nerve regeneration in vivo. Our data indicates a functional switch for the role of Sema3A in PNS neurons where the well-described repulsive role during development changes to a growth promoting effect during adulthood. The high expression of Sema3A in the normal and injured adult corneas could be related to its role as a growth factor.

Semaphorin3A induces nerve regeneration in the adult cornea-a switch from its repulsive role in development

Anatomy of the human corneal innervation.

The central projections of rat trigeminal primary afferent neurons to various “non-trigeminal” areas of the central nervous system were examined by labeling the fibers with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) transported anterogradely from the trigeminal ganglion. This technique produced a clear and comprehensive picture of trigeminal primary afferent connectivity that was in many ways superior to that which may be obtained by using degeneration, autoradiography. cobalt labeling, or HRP transganglionic transport techniques. Strong terminal labeling was observed in all four rostrocaudal subdivisions of the trigeminal brainstem nuclear complex, as well as in the dorsal horn of the cervical spinal cord bilaterally, numerous brainstem nuclei, and in. the cerebellum. Labeling in the ipsilateral dorsal horn of the cervical spinal cord was very dense at C1, moderately dense at C2 and C3, and sparse at C4-C7. Numerous fibers crossed the midline in the medulla and upper cervical spinal cord and terminated in the contralateral pars caudalis and dorsal horn of the spinal cord from C1-C5. The latter axons terminated most heavily in the mandibular and ophthalmic regions of the contralateral side. Extremely dense terminal labeling was observed in the ipsilateral paratrigeminal nucleus and the nucleus of the solitary tract, moderate labeling was seen in the supratrigeminal nucleus and in the dorsal reticular formation, and small numbers of fibers were observed in the cuneate, trigeminal motor, lateral and superior vestibular nuclei, and in the cerebellum. The tatter fibers entered the cerebellum in the superior cerebellar peduncle and projected to the posterior and anterior lobes as well as to the interposed and lateral deep cerebellar nuclei. Most projections in this study originated from fibers in the dorsal part of the spinal tract of V, suggesting a predominantly mandibular origin for these fibers. Projections from the ophthalmic and maxillary divisions, in contrast, were directed mainly to the cervical spinal cord bilaterally, to contralateral pars caudalis, and to certain areas of the reticular formation. In conclusion, this study has demonstrated that somatosensory information from the head and face may be transmitted directly to widespread and functionally heterogeneous areas of the rat central nervous system, including the spinal cord dorsal horn, numerous brainstem nuclei, and the cerebellum. These projections may play important roles in trigeminospinal reflexes (dorsal horn of spinal cord C1-C7), fusion of the sensory maps of the right and left sides of the head (contralateral projections), trigeminovisceral integration (paratrigeminal and solitary), control and integration of oral motor behavior (supratrigeminal and motor V), orofacial reflexes (reticular formation), and the coordination and stabilization of head posture and gaze (cuneate, vestibular, and cerebellum).

Trigeminal primary afferent projections to “non-trigeminal” areas of the rat central nervous system

The peptidergic and serotoninergic innervation of the rat dura mater was investigated by reacting dural wholemounts immunohistochemically with antibodies to calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), and serotonin (5-HT). CGRP and SP innervations of the dura were robust and the patterns of distribution of these neuropeptides were essentially the same. The majority of the fibers were perivascular and distributed to branches of the anterior and middle meningeal arteries and to the superior sagittal and transverse sinuses. Other CGRP/SP fibers appeared to end "free" within the dural connective tissue. NPY-immunoreactive fibers were extremely numerous and also distributed heavily to the branches of the meningeal arteries, the venous sinuses, and to the dural connective tissue. The pattern of NPY innervation resembled in many ways that of CGRP/SP; however, NPY innervation of the sinuses was greater and NPY perivascular fibers supplying the meningeal arteries formed more intimate contacts with the walls of the vessels. The pattern of VIP innervation was, in general, similar to that observed for the three previous neuropeptides; however, the overall density was considerably less. Small to moderate numbers of serotoninergic nerve fibers were observed in some, but not all, of the duras processed for 5-HT. The latter fibers were almost exclusively perivascular in distribution. Dural mast cells were prominently stained in the 5-HT preparations because of their serotonin content. Mast cells were also labeled in a nonspecific fashion in some of the tissues reacted immunohistochemically for neuropeptides; some of them were located in close apposition to passing nerve fibers. This study represents, to our knowledge, the first comprehensive work on the peptidergic and serotoninergic innervation of the mammalian dura mater. The results should increase our understanding of the roles that these fibers play in normal dural physiology and of their potential interactions in the pathogenesis of vascular headache.

Peptidergic and serotoninergic innervation of the rat dura mater.

The methods of transganglionic transport of horseradish peroxidase (HRP) and horseradish peroxidase--wheat germ agglutinin (HRP-WGA) were used to determine the location within the trigeminal ganglion of the primary afferent neurons that innervate the rat central cornea, and the brainstem and spinal cord termination sites of these cells. In each of 18 animals, solutions of HRP or HRP-WGA were applied to the scarified corneal surface and allowed to infiltrate into the corneal epithelium and stroma for 15 minutes. Postmortem examination of the corneal whole mounts from the experimental animals, and of corneas and neural tissues from several control animals, showed that the HRP/HRP-WGA remained confined to the central cornea with no spread into adjacent intra- or extraorbital tissues. HRP-labeled corneal afferent somata were located in the dorsal part of the ophthalmic region of the ipsilateral trigeminal ganglion. The central fibers of the corneal afferent neurons projected very heavily to interstitial nuclei of Cajal in the spinal tract of V at the level of caudal pars interpolaris and rostral pars caudalis, lightly to the pars caudalis/C1 transition zone, and sparsely to the dorsal horn of spinal cord segments C1-C3. The trigeminal main sensory nucleus, pars oralis, the rostral three-fourths of pars interpolaris, and an extensive midregion of pars caudalis were totally devoid of reaction product. Terminal fields in caudal pars caudalis and in the spinal cord dorsal horn were concentrated largely in the outer half of lamina II, with lesser accumulations in lamina I, the deeper half of lamina II, and in lamina III. The present study demonstrates for the first time by means of an anatomical tracing procedure the brainstem termination sites of corneal afferent neurons in the rat. The patchy, discontinuous nature of the corneal afferent projection to the caudal trigeminal brainstem nuclear complex (TBNC), and the total lack of corneal projections to rostral subdivisions of the TBNC, provide an exception to the general rule of trigeminal organization in which most areas of the head and face are represented as continuous columns throughout the rostrocaudal extent of the ipsilateral TBNC.

Carl F. Marfurt

Papers

Corneal nerves: structure, contents and function.

Anatomy of the human corneal innervation.

Trigeminal primary afferent projections to “non-trigeminal” areas of the rat central nervous system

Peptidergic and serotoninergic innervation of the rat dura mater.

Corneal sensory pathway in the rat: a horseradish peroxidase tracing study.