Femtosecond laser cutting of human corneas for the subbasal nerve plexus evaluation.

Assessment of various morphological parameters of the corneal subbasal nerve plexus is a valuable method of documenting the structural and presumably functional integrity of the corneal innervation in health and disease. The aim of this work is to establish a rapid, reliable and reproducible method for visualization of the human corneal SBP using femtosecond laser cut corneal tissue sections. Trephined healthy corneal buttons were fixed and processed using TissueSurgeon-a femtosecond laser based microtome, to obtain thick tissue sections of the corneal epithelium and anterior stroma cut parallel to the ocular surface within approximately 15 min. A near infrared femtosecond laser was focused on to the cornea approximately 70-90 µm from the anterior surface to induce material separation using TissueSurgeon. The obtained corneal sections were stained following standard immunohistochemical procedures with anti-neuronal ß-III tubulin antibody for visualization of the corneal nerves. Sections that contained the epithelium and approximately 20-30 µm of anterior stroma yielded excellent visualisation of the SBP with minimal optical interference from underlying stromal nerves. In conclusion, the results of this study have demonstrated that femtosecond laser cutting of the human cornea offers greater speed, ease and reliability than standard tissue preparation methods for obtaining high quality thick sections of the anterior cornea cut parallel to the ocular surface.

Comparative quantitative assessment of the human corneal sub-basal nerve plexus by in vivo confocal microscopy and histological staining.

PurposeThis study was designed to compare and contrast quantitative data of the human corneal sub-basal nerve plexus (SBP) evaluated by two different methods: in vivo confocal microscopy (IVCM), and immunohistochemical staining of ex vivo donor corneas.MethodsSeven parameters of the SBP in large-scale IVCM mosaicking images from healthy subjects were compared with the identical parameters in ex vivo donor corneas stained by ß-III-tubulin immunohistochemistry. Corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), average weighted corneal nerve fiber tortuosity (CNFTo), corneal nerve connection points (CNCP), average corneal nerve single-fiber length (CNSFL), and average weighted corneal nerve fiber thickness (CNFTh) were calculated using a dedicated, published algorithm and compared.ResultsOur experiments showed significantly higher values for CNFL (50.2 vs 21.4 mm/mm2), CNFD (1358.8 vs 277.3 nerve fibers/mm2), CNBD (847.6 vs 163.5 branches/mm2), CNFTo (0.095 vs 0.081 µm-1), and CNCP (49.4 vs 21.6 connections/mm2) in histologically staining specimens compared with IVCM images. In contrast, CNSFL values were higher in IVCM images than in histological specimens (32.1 vs 74.1 µm). No significant difference was observed in CNFTh (2.22 vs 2.20 µm) between the two groups.ConclusionsThe results of this study have shown that IVCM has an inherently lower resolution compared with ex vivo immunohistochemical staining of the corneal SBP and that this limitation leads to a systematic underestimation of several SBP parameters. Despite this shortcoming, IVCM is a vital clinical tool for in vivo characterization, quantitative clinical imaging, and evaluation of the human corneal SBP.

Morphology and neurochemistry of canine corneal innervation.

PURPOSE: To determine the architectural pattern and neuropeptide content of canine corneal innervation. METHODS: Corneal nerve fibers in normal dog eyes were labeled immunohistochemically with antibodies against protein gene product (PGP)-9.5, calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), and tyrosine hydroxylase (TH). Relative innervation densities and distribution patterns for each fiber population were assessed qualitatively by serial line-drawing reconstructions and quantitatively by computer-assisted analyses. RESULTS: More than 99% of all corneal PGP-9.5-immunoreactive (IR) nerves contained both CGRP and SP, approximately 30% contained TH, and none contained VIP. Distribution patterns of corneal PGP-9.5-, CGRP-, SP-, and TH-IR nerves were indistinguishable, except that TH-IR fibers were absent from the corneal epithelium. Morphologically, canine corneal innervation consisted of a rich anterior stromal plexus, divided on the basis of morphologic criteria into anterior and posterior levels, and a rich epithelial innervation, characterized by large numbers of horizontally oriented, basal epithelial "leash" formations. Leash axons in all quadrants of the corneal epithelium oriented preferentially toward a common locus in the perilimbal cornea. CONCLUSIONS: The results of this study demonstrate for the first time the detailed architectural features, distinctive basal epithelial leash orientations, and peptidergic content of canine corneal innervation. The normal innervation pattern described in this study will provide other investigators with essential baseline data for assessing corneal nerve alterations in canine patients with spontaneous chronic corneal epithelial defects (SCCED) and other ocular diseases or injuries.

Spontaneous chronic corneal epithelial defects (SCCED) in dogs: clinical features, innervation, and effect of topical SP, with or without IGF-1.

PURPOSE: To delineate the clinical features and alterations in innervation and substance P (SP) content in spontaneous chronic corneal epithelial defects (SCCED) in dogs and to conduct a preliminary investigation evaluating the efficacy of topical SP, with or without insulin-like growth factor (IGF)-1, in the treatment of this disorder. METHODS: Complete ophthalmic examinations, including Cochet-Bonnet aesthesiometry, were performed in 45 canine patients that had spontaneous corneal epithelial defects of at least 3 weeks' duration and with no identifiable cause. Eighteen patients had superficial keratectomies performed, and the corneal nerves were labeled immunohistochemically with antibodies against protein gene product (PGP)-9.5, SP, vasoactive intestinal peptide (VIP), and tyrosine hydroxylase (TH). Relative fiber densities were assessed qualitatively and quantitatively. Corneal epithelial cell and tear SP contents were determined in affected and normal dogs by an enzyme immunoassay. A preliminary open-label treatment trial of topical SP, with and without IGF-1, was conducted in 21 dogs. RESULTS: The duration of the erosion before admittance into the study was a mean of 9.22 weeks (range, 3-52). The average patient was middle aged (mean, 9.25 +/- 1.85 years [SD]); no sex predisposition of the disease was identified. Boxers, golden retrievers, and keeshonds were overrepresented when compared with the normal hospital population. Corneal sensation was normal. Marked alterations in corneal innervation were identified in affected dogs with abnormal increased SP and calcitonin gene-related peptide (CGRP)-immunoreactive nerve plexuses identified surrounding the periphery of the epithelial defect. The SP content of epithelial cells surrounding the defect increased, whereas the tear SP content remained unchanged. Of the canine patients treated with SP, with or without IGF-1, 70% to 75% had complete healing of the defect. CONCLUSIONS: This idiopathic spontaneous corneal disease in dogs shares clinical features with chronic epithelial defects in humans. The presence of marked alterations in peptidergic innervation and positive response to topical therapy with SP suggest that SP plays a critical role in corneal wound-healing processes.

Effect of norepinephrine on proliferation, migration, and adhesion of SV-40 transformed human corneal epithelial cells.

PURPOSE: To determine the ability of norepinephrine to modulate proliferation, adhesion, and migration of SV-40 transformed human corneal epithelial cells. METHODS: Assays were performed using SV-40 transformed human corneal epithelial cells. For proliferation assays, cells were plated in 96-well plates coated with fibronectin and collagen (FNC). A dose-response curve was generated for norepinephrine in concentrations of 100 nM-100 microM. The cell number in each well was evaluated using the fluorochrome Calcein AM (an intracellular esterase cleavage substrate), and fluorescence was determined using an automated fluorescent plate reader. For cell adhesion, 25 x 10(-3) cells were plated onto FNC-coated 96-well plates, incubated in 10 nM-100 microM norepinephrine for 90 min, gently irrigated, and the remaining adherent cells quantitated. Cell migration was measured using blind-well migration chambers with a 10-microm pore size and FNC-coated filters. Cells (250 x 10(3)) were added to the upper chamber, incubated for 18 h in the presence of factors, after which time the cells that had migrated through the filter were quantitated. The toxicity of norepinephrine was evaluated using a standard Live/Dead assay employing the combined fluorochromes of ethidium homodimer (to indicate dead cells) and Calcein AM (to indicate viable cells). Varying concentrations of norepinephrine were added, and the cells incubated for 3 h and the fluorometric assay performed. RESULTS: Norepinephrine stimulated corneal epithelial cell proliferation and migration over a wide range of concentrations. It did not modulate cell adhesion and demonstrated cell toxicity only at the highest (supraphysiologic) concentration tested. CONCLUSIONS: Norepinephrine is normally found in the cornea and may be important in the maintenance of normal corneal homeostasis and in wound-healing processes.

Parasympathetic innervation of the rat cornea.

The mammalian cornea receives a dense sensory innervation and a modest sympathetic innervation. The purpose of the current study was to determine if the rat cornea is also innervated by parasympathetic nerves. In the first set of experiments, unilateral combined sympathetic and sensory ocular denervations were performed in adult rats by surgical removal of the superior cervical ganglion and intracranial transection of the trigeminal ophthalmomaxillary nerve. Completeness of the denervation procedure was verified postmortem by a variety of macroscopic and immunohistochemical methods. Five to twelve days later, the corneas were serially sectioned tangential to the ocular surface and processed immunohistochemically with antibodies against the pan-neuronal markers, protein gene product 9.5 (PGP-9.5) and peripherin. In every animal a small, but constant, population of corneal and limbal immunoreactive fibers were unaffected by the surgical denervations and were concluded to derive from parasympathetic ganglia. In the second set of experiments, the origins of the rat corneal innervation were determined by applying the neuroanatomical tracer, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to the central cornea. Two to four days later, the trigeminal, superior cervical, ciliary, accessory ciliary and pterygopalatine ganglia were sectioned and analysed for the presence of HRP-labeled neurons. Examination of the corneal application site and associated ocular tissues revealed no evidence of tracer spread into neighbouring structures. Small numbers (0-6 per animal) of HRP-labeled neurons were observed in the ipsilateral ciliary and accessory ciliary ganglia of most animals. The results of these carefully controlled studies provide strong anatomical evidence of a modest parasympathetic innervation of the rat cornea.

Peptidergic innervation of the rat cornea.

Corneal nerves regulate corneal epithelial integrity, proliferation, and wound healing. The mechanisms by which the nerves mediate their effects remain poorly understood; however, the release of biologically active neuropeptides has been hypothesized. The purpose of the current investigation was to determine the relative densities, distribution patterns, and origins of rat corneal nerves containing each of eight different neuropeptides, calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), neuropeptide Y (NPY), methionine-enkephalin (M-ENK), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), and cholecystokinin (CCK). In the first set of experiments, immunohistochemical demonstrations of the above neuropeptides were performed on free-floating corneal sections cut tangential to the corneal surface. The results showed that six of the peptides, CGRP, SP, GAL, NPY, M-ENK, and VIP were present in rat corneal nerves. The innervation patterns of corneal nerves containing each of these six peptides were then documented by mapping all fibers in serial sections from select corneal quadrants onto a series of line drawings by using a drawing tube. In the second set of experiments, the origins of the corneal peptidergic nerve fibers were determined by selective ocular denervations. Unilateral combined sensory and sympathetic ocular denervations or unilateral sympathetic ocular denervations were performed in adult rats by transecting the ophthalmomaxillary nerve and/or removing the superior cervical ganglion. After 5-7 days, each of the ipsilateral corneas was sectioned and processed immunohistochemically for the presence of one of the six peptides found in experiment one, and the fibers that survived the ocular denervations were plotted onto line drawings. Ocular denervations revealed that corneal peptidergic nerves have sensory (CGRP, SP, and GAL), sympathetic (NPY), and parasympathetic (GAL, NPY, M-ENK, and VIP) origins. The results of this investigation have shown that the peptidergic innervation of the rat cornea is more extensive and complex than previously reported. This is the first investigation to show the presence of GAL in the rat cornea, and the first to demonstrate the presence of NPY-, VIP-, and M-ENK-IR nerve fibers in the cornea of any species.

Topical substance P and corneal epithelial wound closure in the rabbit.

PURPOSE: The authors determined the effect of topically applied substance P (SP) on the rate of corneal epithelial wound closure in the rabbit. METHODS: Uniform circular lesions, 6.5 mm in diameter, were made bilaterally in the corneal epithelium of 24 rabbits using N-heptanol. Substance P was applied repeatedly to one eye, and the SP1-7 fragment was applied to the contralateral (control) eye until wound closure was obtained. Three concentrations of peptide solution (5 x 10(-5) M, 5 x 10(-4) M, and 5 x 10(-3) M) were tested in separate groups of eight animals each. An additional eight animals received topical applications (5 x 10(-7) M) of the neurokinin-1 (NK1)-specific SP receptor antagonist CP-99,994-01 or its ineffective enantiomer CP-100,263-01. The mean rates of wound healing for each group of experimental and control eyes were determined by linear regression and analyzed by analysis of variance. RESULTS: There were no statistically significant differences in the mean rates of wound closure (range, 0.083 to 0.106 mm/hour) between experimental- and control-treated corneas for any of the four groups tested. CONCLUSIONS: The topical application of SP or its NK1 receptor antagonist has no significant effect on the rate of corneal epithelial wound closure in the rabbit.

Sympathetic stimulation of corneal epithelial proliferation in wounded and nonwounded rat eyes.

PURPOSE: To determine the effect of ocular sympathetic nerves on corneal epithelial proliferation in the rat. METHODS: Osmotic pumps filled with bromodeoxyuridine (BrdU) were implanted subcutaneously in adult rats to label corneal epithelial cells entering the S-phase of the cell cycle during a 24-hour period. Corneas in some animals were wounded with n-heptanol. Several days or weeks before pump implantation, animals were subjected to either unilateral superior cervical ganglionectomy (SCGectomy), unilateral transection of the cervical sympathetic trunk (sympathetic decentralization), bilateral SCGectomy plus unilateral topical norepinephrine administration, or unilateral SCGectomy plus systemic capsaicin administration. Differences in BrdU-labeling indices between experimental and control eyes in each group were determined from cell counts on paraffin sections. RESULTS: Superior cervical ganglionectomy and sympathetic decentralization significantly decreased epithelial proliferation in nonwounded and wounded corneas. Topical applications of norepinephrine to eyes that had been deprived of their sympathetic innervation completely reversed the antiproliferative effect of ocular sympathectomy. Systemic administration of the neurotoxin capsaicin, in conjunction with unilateral SCGectomy, did not alter the proliferative rate; comparison was made to animals that received unilateral SCGectomy only. CONCLUSIONS: Ocular sympathetic nerves stimulate rat corneal epithelial proliferation under normal physiological conditions and during corneal wound healing. The promotion of DNA synthesis by these nerves occurs independently of functional interactions with capsaicin-sensitive, ocular sensory nerves and appears to be related, at least in part, to the release of norepinephrine.

Local anesthetic effects in the presence of chronic osteomyelitis (necrosis) of the mandible: implications for localizing the etiologic sites of referred trigeminal pain.

The aims of this study were: (1) to demonstrate how reproducible variations in incomplete anesthesia of the inferior alveolar nerve can be used as a guide to locate the etiologic sites of referred trigeminal pain emanating from the mandible; (2) to describe the salient histopathologic features of a lowgrade, nonsuppurative osteomyelitis seen in this patient population. Forty-six patients with idiopathic facial pain were subjected to a specific protocol of local anesthetic injections to sequentially block branches of the mandibular nerve to determine the effects on his/her pain. If this significantly reduced or altered the pain on three separate appointments, then exploratory surgery was conducted near identified zones of unanesthetized gingiva. Blocking (92%), bridging (4%), and divergence (4%) were observed patterns of anesthetic resistance of the mucogingival tissues used to categorize the incomplete anesthesia. A 100% correlation was found between the identified zones of unanesthetized gingiva and the discovery of intramedullary pathology. Medullary fibrosis with ischemic and degenerative changes in the cancellous bone were common findings, along with chronic inflammatory cell infiltrates and clusters of lymphocytes. It is concluded that Ratner's method of diagnostic anesthesia be implemented when searching for occult pain producing pathology of the jaws.

The effect of diabetes on neuropeptide content in the rat cornea and iris.

PURPOSE: To determine the effect of diabetes mellitus on the levels of substance P (SP), calcitonin gene-related peptide (CGRP), and vasoactive intestinal polypeptide (VIP) in the rat cornea and iris. METHODS: Corneas and irides from control and diabetic rats were processed for neuropeptide radioimmunoassay 3 months after induction of diabetes with streptozotocin. Corneas and irides also were processed for SP and CGRP immunohistochemistry and were evaluated qualitatively. RESULTS: The radioimmunoassay data revealed no significant differences in either the content or concentration of SP, CGRP, and VIP between control and diabetic corneas. In contrast, iridial levels of CGRP and SP were significantly increased by 38% and 256%, respectively, in the diabetic animals. Iridial VIP levels were unchanged in the diabetic versus control groups. Immunohistochemical demonstrations of corneal and iridial SP- and CGRP-immunoreactive fiber plexuses were indistinguishable on the basis of purely qualitative criteria. CONCLUSIONS: The results of this study have demonstrated a target- and peptide-specific effect of short-term diabetes on SP and CGRP expression in ocular nerves of the anterior eye segment. The absence of demonstrable changes in corneal neuropeptide levels argue against the theory that corneal abnormalities seen in clinical diabetes are caused, in part, by deficits in synthesis or axonal transport of "trophic" peptides in corneal sensory nerves. In contrast, elevated iridial SP and CGRP levels may be responsible for reported clinical deficits in pupillary diameter regulation.

Immunohistochemical localization of tyrosine hydroxylase in corneal nerves.

The sympathetic innervation of the mammalian cornea is thought to play an important role in the regulation of epithelial ion transport, mitogenesis, and wound healing following corneal injuries. Anatomically, the three-dimensional organization and relative density of corneal sympathetic innervation in many species remains inadequately described. In the present study, the sympathetic innervation of five different mammals (guinea pig, rat, mouse, hamster, and human) was studied in corneas sectioned parallel to the main axis of fiber orientation by labeling the fibers immunohistochemically with antiserum against tyrosine hydroxylase and an avidin-biotin-diaminobenzidine technique. The results showed that each species displayed a distinctive pattern and density of tyrosine hydroxylase immunoreactive (TH-IR) corneal innervation that was unique to that species. The overall level of TH-IR innervation was highest in the guinea pig, moderate in the human, hamster, and rat, and lowest in the mouse. In all species examined, TH-IR nerves were most numerous in the corneoscleral limbus where they either formed intimate associations with blood vessels or coursed through the connective tissue matrix apparently unrelated to vascular elements. Other TH-IR nerves entered the cornea proper in radially directed stromal nerve bundles to give rise to subepithelial plexuses of varying complexity. Occasional intraepithelial penetrations were observed in the guinea pig, human, and rat. Removal of the superior cervical ganglion resulted in the total loss of TH-IR staining from guinea pig and hamster corneas and in the substantial but incomplete loss of TH-IR staining from rat and mouse corneas, thus demonstrating their predominantly sympathetic origin. Combined sympathetic and sensory ocular denervation in the rat eliminated almost all corneal and limbal TH-IR immunostaining, thus suggesting a minor TH-IR sensory component in this species. In agreement with this conclusion, small numbers of TH-IR sensory neurons and an abundance of TH-IR fibers were observed in the trigeminal ganglia of the rat and guinea pig. Removal of the rat main ciliary ganglion resulted in the loss of additional TH-IR fibers from the chamber angle and iris, thereby confirming a partial parasympathetic contribution to the rat iridial TH-IR innervation. Following unilateral removal of the superior cervical ganglion in rats and guinea pigs, the contralateral cornea contained increased numbers of TH-IR nerves, suggesting an upregulation of tyrosine hydroxylase (TH) expression in some contralateral axons.(ABSTRACT TRUNCATED AT 400 WORDS)

Sensory and sympathetic nerve sprouting in the rat cornea following neonatal administration of capsaicin.

Corneal sensory and sympathetic nerves exert opposing actions on corneal mitogenesis and wound healing. The mechanisms by which these nerves exert their actions are unknown; however, the release of axonally transported neuropeptides has been postulated. In the present study, we investigated changes in innervation densities of calcitonin gene-related peptide (CGRP-) and tyrosine hydroxylase (TH-)immunoreactive (IR) nerves of the rat cornea following neonatal capsaicin administration, and the relationships between these changes and the development of neuroparalytic keratitis. Newborn rats were injected with capsaicin on each of the first 3 days of life. Forty-eight hours after the last injection, corneal CGRP immunostaining had totally disappeared from the cornea, whereas TH immunostaining was relatively unaffected. Over the next several weeks, a dramatic reinnervation of the cornea took place. By 6-8 weeks both the CGRP- and TH-IR corneal innervation density in the capsaicin-treated animals exceeded that of age-matched control or normal animals; that is, the corneas had become "hyper-reinnervated." The pattern of innervation that returned was grossly abnormal and was characterized by the presence of a bizarre subepithelial plexus of fine stromal sprouts; an abundance of myelinated axons; and complex, atypical, epithelial leash morphologies. Retrograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) from the central cornea in control and capsaicin-treated adult animals labeled an average of 143 and 47 trigeminal ganglion cells, respectively (with mean diameters of 25.7 +/- 0.49 microns and 34.3 +/- 0.72 microns), suggesting a 67% decrease in corneal afferent neurons in the capsaicin-treated animals. Transection of the ophthalmomaxillary nerve in adult capsaicin-treated animals completely eliminated corneal CGRP-IR staining, and extirpation of the superior cervical ganglion resulted in the loss of 70-80% of corneal TH-IR nerves, thus demonstrating the sensory and predominantly sympathetic origins, respectively, of these fiber populations. Chronic keratitis and neovascularization developed in the capsaicin-treated animals by approximately 3 weeks of age, achieved a maximum intensity between 4 and 6 weeks, and showed some gradual improvement thereafter. However, the keratitis never completely disappeared, even after 13 months. In conclusion, these data show that corneal sensory (CGRP-IR) and sympathetic (TH-IR) nerve fibers undergo extensive sprouting following partial corneal sensory denervation with the neurotoxin capsaicin. However, the resultant "hyper-reinnervation" is morphologically abnormal and, for reasons unknown, functionally incapable of preventing or totally reversing the keratitis.

Calcitonin gene-related peptide and corneal innervation: a developmental study in the rat.

The development of calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerves was studied in neonatal and adult rat corneas stained immunohistochemically according to an avidin biotin peroxidase procedure. At birth, rat corneas already contained dense plexuses of CGRP-LI nerve fibers. Most of the nerves entered the cornea in 12-15 prominent stromal nerve bundles located at regular intervals around the circumference of the cornea. Fibers in these bundles entered the epithelium approximately midway between the limbus and the center of the cornea and supplied extensive central and pericentral areas of the tissue. In addition, smaller numbers of axons entered the cornea individually and in small fascicles located in between the larger bundles and supplied mainly peripheral territory. In the epithelium, the CGRP-LI nerves formed a complex, highly anastomotic meshwork that ramified uniformly throughout central and peripheral areas of the tissues. Fibers in the plexus gave origin to numerous short, stout terminal axons that extended into the adjacent epithelium in all directions with no preferred orientation. During the first week of neonatal life, several changes in CGRP-LI innervation occurred: 1) the innervation density of the central and pericentral cornea increased relative to the peripheral cornea; 2) intraepithelial axons became progressively longer, increased in branching complexity, and oriented preferentially towards the center of the cornea; and 3) a dense innervation of the corneoscleral limbus and, in particular, the branches of the marginal artery, developed. Midway through the second week of life, immature versions of corneal epithelial "leashes," the dominant feature of the adult corneal innervation, were first observed. Over the next 10 days, the leash formations in the central and pericentral cornea gradually became more complex and gave rise to greater numbers of terminal axons, compared to developing leashes in the peripheral cornea. The mature pattern of corneal CGRP-LI innervation was reached on day 21 and remained constant (except for compensatory growth-related elongation of axons) for at least the first 6 months of life. Transection of the ophthalmomaxillary nerve or neonatal administration of the sensory neurotoxin capsaicin resulted in the total loss of CGRP-LI staining from the cornea. In contrast, removal of the superior cervical ganglion had no effect on corneal CGRP-LI staining. The extraordinary density and complexity of the CGRP-LI innervation of the rat cornea demonstrated at all stages of development in this study suggests that these nerves may play important roles in corneal sensory, reflex, and trophic functions.

Sensory innervation of the rat kidney and ureter as revealed by the anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) from dorsal root ganglia.

The sensory innervation of the rat kidney and ureter was investigated in wholemount preparations and sectioned materials by labeling the afferent nerve fibers with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) transported anterogradely from dorsal root ganglia. Labeled fibers were seen in large numbers in the ureter and in the lining of the renal pelvis, where they were located in the adventitia, smooth muscle, subepithelial connective tissue, and epithelium. Most of the fibers in the ureter and ureteropelvic junctional zone traveled parallel to the long axis of the organ. In contrast, fibers in the widest part of the funnel-shape renal pelvis were oriented predominantly in a circumferential fashion. Many of the pelvic afferents were extremely fine and appeared to terminate as free nerve endings. Modest networks of labeled axons were also observed around branches of the renal artery; the greatest innervation was supplied to the distal portions of the interlobar arteries and to the arcuate arteries. Only single axons were observed around the interlobular arteries, and very few fibers were seen around afferent arterioles or near glomeruli. In contrast to the arteries, branches of the renal vein were relatively sparsely innervated. Occasional labeled fibers entered the renal cortex and formed intimate associations with renal tubules; however, the vast majority of renal tubular elements were not contacted by labeled sensory fibers. Labeled fibers were never observed in the renal medulla or in the papilla. The present study represents the first time that the sensory innervation of the kidney and ureter has been investigated by using a highly specific anterograde nerve tracing technique. The pattern of innervation demonstrated here reveals an anatomical configuration of ureteral and renal pelvic sensory nerves consistent with a role in detection of ureteral and pelvic pressure and chemical changes and a renal vascular sensory innervation that may monitor changes in renal arterial and venous pressure and chemical content. Still other renal afferent nerve endings may signal renal pain.

Peptidergic and serotoninergic innervation of the rat dura mater.

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.

Trigeminal primary afferent projections to "non-trigeminal" areas of the rat central nervous system.

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 latter 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Sympathetic innervation of the supratentorial dura mater of the rat.

The origin, density, and distribution of sympathetic nerve fibers in the supratentorial dura mater of the rat were examined in detail in the current study by using wheat germ agglutinin horseradish peroxidase (WGA-HRP) retrograde tracing procedures, glyoxylic acid-induced fluorescence, and dopamine beta-hydroxylase (DBH) immunocytochemical staining of dural whole mount preparations. Application of WGA-HRP to the superior sagittal sinus and adjacent areas of the supratentorial dura mater labeled numerous neurons in each of the left and right superior cervical ganglia. Glyoxylic acid and DBH immunocytochemical staining of fixed dural whole mount preparations revealed prominent plexuses of sympathetic nerves about the middle meningeal artery and its branches, about the superior sagittal and transverse sinuses, and "free" within the dura mater, i.e., apparently unassociated with any vasculature. Significantly, in all of these areas, the density of sympathetic innervation revealed in this study was considerably greater than that previously demonstrated by other workers. An impressive population of mast cells also was observed within the dura mater of the glyoxylic acid-treated preparations. The majority of these cells were perivascular; however, a significant number were also present within the dura unrelated to the vasculature, and occasional cells were seen in close apposition to fluorescent sympathetic nerve fibers. Taken together, the identification of a robust sympathetic plexus and prominent mast cell population associated with a dura mater that also receives significant sensory projections from the trigeminal system raises interest regarding the functional interactions of these elements. These observations warrant further consideration regarding their role in the pathogenesis of vascular headache and head pain.

Demonstration of physiological barrier between pulpal odontoblasts and its perturbation following routine restorative procedures: a horseradish peroxidase tracing study in the rat.

Vascular injection of the macromolecular tracer, horseradish peroxidase (HRP), was used to study the permeability of the odontoblast cell layer in developing and mature rat molar teeth, and to investigate the effect of cavity preparations on the permeability of this epithelioid cell layer in adult animals. HRP injected into the vascular system of normal animals 28 days of age and older was localized histochemically (from 5 to 90 min after injection) throughout the extracellular spaces of the maxillary dental pulps; however, the tracer did not penetrate beyond the tight junctions at the apical region of the odontoblast cell layer, and was absent from the predentin and dentin. In contrast, HRP injected into very young neonatal animals (e.g., day 3) resulted in free passage of HRP between odontoblasts and into the overlying predentin and dentin. When Class V cavities had been prepared in adult maxillary molars after HRP was injected into the blood stream, HRP reaction product penetrated the predentin and dentin immediately beneath the cavity preparation; however, adjacent, untraumatized areas of predentin and dentin in the operated teeth were devoid of reaction product. These results provide evidence that: (1) a physiological barrier develops between the distal segments of odontoblast cell bodies in normal rat molar teeth between days 15 and 28 of postnatal life, and this barrier prevents the passage of macromolecules from the pulp into the predentin and dentin; and (2) this barrier is perturbed following routine restorative procedures in adult animals.

Origins of the renal innervation in the primate, Macaca fascicularis.

The origins of the renal efferent and afferent nerves in 5 cynomolgus monkeys (Macaca fascicularis) were studied by using the retrograde transport of horseradish peroxidase (HRP) and horseradish peroxidase-wheat germ agglutinin (HRP-WGA). The cut ends of the right renal nerves were soaked for 30-45 min in solutions consisting of 15% HRP and 1% HRP-WGA. Three or four days later the animals were killed and the tissues examined for the presence of retrogradely labeled neurons, HRP-filled cells were observed, with rare exceptions, only in ganglia ipsilateral to the side of tracer application. Renal efferent neurons (4648-14565 cells per animal) were found in relatively equal numbers in prevertebral and paravertebral (sympathetic chain) ganglia. Labeled prevertebral cells were distributed among the renal (52%), aorticorenal (32%) and superior mesenteric (16%) ganglia, whereas labeled paravertebral neurons were mainly located in chain ganglia T11-L3, with 94% of these located in L1-3. Labeled renal sensory neurons (31-543 per animal) constituted less than 5% of all labeled cells and were found in ipsilateral dorsal root ganglia T10-L3, with (80%) in T12 and L1. The labeled sensory neurons ranged from 18-64 microns in diameter (X = 32.4 microns). With the exception of a single cell in one animal, no labeled neurons were observed in the nodose ganglia. Many parallels were observed between the organization of the renal plexuses of macaques and humans, suggesting the utility of the non-human primate as an experimental model for functional studies of renal innervation in humans.