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. 2005 Mar;206(3):217–224. doi: 10.1111/j.1469-7580.2005.00390.x

Re-evaluation and quantification of the different sources of nerve fibres supplying the rat eye

Carlo Cavallotti 1, Alessandro Frati 3, Paolo Sagnelli 1, Nicola Pescosolido 2
PMCID: PMC1571470  PMID: 15733292

Abstract

The denervation and/or the removal of peripheral nerve ganglia are useful surgical techniques for studying the source and distribution of peripheral nerves in all organs, including the eye. The amount and distribution of the remaining nerve fibres supplying the eye (after sectioning of various types of nervous fibres and/or removal of nerve ganglia) were evaluated in the rat. Male Sprague–Dawley rats were anaesthetized and one or more of the following nervous tissues were removed: superior cervical ganglion, main ciliary ganglion, pterygopalatine ganglion, trigeminal ganglion and the ophthalmic-maxillary nerve. In some animals, chemical sympathectomy was performed by administration of 6-OH dopamine. The eyes were cut in serial sections, but only three regions (cornea, iris and choroid) were harvested and submitted for various nerve fibre staining techniques. The results were quantified and statistically analysed. Superior cervical ganglionectomy and/or chemical sympathectomy induced the destruction of almost all the catecholaminergic nerve fibres in the three examined regions of the rat eye. Removal of the ciliary ganglion (partial parasympathectomy) caused the destruction of about 60% of the cholinergic nerve fibres of the same regions of the rat eye, while subtotal parasympathectomy destroyed about 80% of the cholinergic nerve fibres. Surgical transsection of the ophthalmo-maxillary nerve or the removal of the trigeminal ganglion led to a degeneration of almost all sensitive nerve fibres of the three examined regions of the rat eye. The denervation experiments confirmed the presence of the different types of nerve fibres (sympathetic, parasympathetic and sensitive) in the three studied structures of the rat eye.

Keywords: eye, nerve section, nervous ganglia removal, ocular peripheral ganglia, parasympathectomy, sympathectomy

Introduction

It is more than a century since the well-elaborated intrinsic nervous system supplying the cornea was described in the human eye (Müller, 1859; Krause, 1861). In recent decades the nerves of the eye, particularly the nerves of the cornea, iris and choroid, were more specifically characterized because of their neurotransmitter profiles, including not only human but other mammalian and avian eyes (Stone et al. 1987; Bill et al. 1991; Denis et al. 1993). Taking the results of all of these studies together, it becomes evident that there are large variations in the transmitters and/or receptors in the nerve fibres of the eye between all species studied (Cavallotti et al. 1998a,b, 2000, 2002).

In the human eye, numerous neuropeptides have been localized to the vascular walls throughout the choroid, including calcitonin gene-related peptide (CGRP; Stone, 1986a; Uusitalo et al. 1989), substance P (SP; Miller et al. 1982; Stone & Kuwayama, 1985), vasoactive intestinal peptide (VIP; Miller et al. 1982; Uusitalo et al. 1984; Stone, 1986b; Flügel et al. 1994; May et al. 2004), neuropeptide Y (NPY; Stone, 1986b) and nitric oxide synthase (nNOS; Bergua et al. 1993; Flügel et al. 1994; Alm et al. 1995; May et al. 2002). Unfortunately, the neurotransmitters cannot be assigned to the single peripheral ganglia; for example, tyrosin hydroxylase (TH)-positive neurons were found in the superior cervical and ciliary ganglia (Kirch et al. 1995), while nNOS-positive neurons were located in the ciliary, trigeminal and pterygopalatine ganglia. Owing to the mixture of neurotransmitters, a clear correlation of the stained peripheral nerve fibres with the corresponding peripheral ganglion is difficult, and a true connection can only be demonstrated by tracing or denervation studies. In the monkey, denervation studies were performed by Gordon Ruskell on the connections of facial nerve fibres and choroid innervation (Ruskell, 1985a,b). As experiments with monkeys are restricted to small numbers, the rat became the most established animal model for this type of study. It has been shown in the rat that the choroid receives a dense sympathetic innervation from the superior cervical ganglion (Kooster et al. 1996) and a dense nitrergic, parasympathetic innervation from the pterygopalatine ganglion (Yamamoto et al. 1993). However, other authors describe only one type of nerve in the various structures of the eye (Roth & Richardson, 1969). Therefore, it is unclear which nerve fibres are represented in each nerve for the different functional groups.

Our experiments in rat eyes were performed to evaluate the distribution of the different types of nerve fibres (sympathetic, parasympathetic and/or sensitive) by means of the nerve fibre transsection technique and also to give correlating data on the different groups of nerve fibres localized in the cornea, iris and choroid.

Materials and methods

Experimental procedures

Forty male Sprague–Dawley rats (12 months old) were used. All experiments were performed in accordance with the Declaration of Helsinki and were approved by the Ethics Committee of our University.

Under general anaesthesia, induced with an intraperitoneal injection of a saline solution containing 4 mg kg−1 body weight penthotal sodium (Abbott), the animals were operated by removal of one or more of those peripheral ganglia that constitute the major sources of ocular nerve fibres (Melby & Altman, 1974). In each experimental group subjected to surgical procedures, the left side was left untouched, for purposes of comparison, while the right side was operated (treated). In further experimental procedures, both sides (treated and untreated) were compared. Moreover, to verify the possible effect of surgical trauma, six animals were sham operated and these served as a control group. Finally, several post-operative examinations were performed to confirm the completeness of the surgical procedures.

Animals were allowed to survive for 7 days, a time period known to be sufficient for degeneration of all nerve fibres (Roth & Richardson, 1969). At that time the animals were killed by terminal anaesthesia and the eyes enucleated. The surgical suture (related to the first operation of nerve transsection or of ganglion removal) was then reopened and the proximal and distal nerve stumps were drawn and examined under a surgical microscope for evidence of intact (non-cut) nerve fibres. Some of the stumps were drawn and examined under a light microscope (after their inclusion and staining) for evidence of intact (normal stained) nerve fibres. The whole length of the stumps was dissected, drawn and studied for the examination of any a-topically placed ganglion neurons. The stump of the nerve or of the removed ganglion was exposed and critically examined under surgical or light microscopes to confirm the completeness of the nerve transsection or ganglion removal.

Surgical sympathectomy

In eight rats, the unilateral superior cervical ganglion (SCG) was removed as described by Ehinger et al. (1969). The eyelids of the homolateral eye were sutured together to inhibit the development of a neuroparalytic keratitis.

Nerve fibres that disappeared following SCG extirpation were considered sympathetic in nature. By contrast, nerve fibres that survived after SCG extirpation were considered either sensory or cholinergic. To ensure the efficiency of the surgical procedure, the iris from both untreated and sympathectomized animals was exposed to formaldehyde vapours (Falck et al. 1962); the iris of sympathectomized animals appeared to be deprived of fluorescent catecholaminergic nerve fibres. Moreover, the efficiency of ganglionectomy was also demonstrated by the appearance of a homolateral ptosis.

Chemical sympathectomy

Destruction of all sympathetic nerve fibres was achieved by means of chemical sympathectomy, following and partially modifying the guidelines proposed by Angeletti & Levi-Montalcini (1970) and by Johnson (1980).

Eight rats received an intraperitoneal injection of 6-OH dopamine in physiological solution with 0.5% ascorbic acid as solvent, in doses of 100 mg kg−1 day−1 for 3 days. This treatment induced the destruction of almost all catecholaminergic nerve fibres after 3–5 days.

Surgical minor parasympathectomy

A partial cholinergic denervation of the right eye was performed in six rats by surgical removal of the major ciliary ganglion (MCG) using a lateral orbital approach as described by Marfurt & Ellis (1993). The ganglion was identified near the bifurcation of the inferior branch of the oculomotor nerve. This inferior branch was entirely removed together with the ganglion by means of microscissors. This surgical procedure induced only a partial ocular parasympathectomy because other cholinergic nerve fibres arise from accessory ciliary ganglia located as a plexus in the optic nerve sheath (Kuwayama et al. 1987) and from the pterygopalatine ganglion (Ten Tusscher et al. 1990).

Surgical major parasympathectomy

It is imposible to obtain a total parasympthectomy in rat eye. A major parasympathectomy was performed in the right eye of six rats by surgical removal of the MCG, accompanied by surgical removal of the pterygopalatine ganglion (Ten Tusscher et al. 1990). This surgical procedure induced an almost subtotal degeneration of the parasympathetic nerve fibres and is called major parasympathetomy.

Section of sensitive ocular nerve fibres

Sensory denervation of the eye was performed by transecting, in six rats, the homo-lateral ophthalmo-maxillary nerve (OMN) that arises from the Gasserian ganglion and lies in a depression in the sphenoid bone. The OMN was cut in correspondence with the foramen orbitorotundum using a lateral orbital approach as described by Marfurt & Ellis (1993) for the identification of the bifurcation of the oculomotor nerve. In an immediately posterolateral side (2 mm) it was possible to identify and cut the OMN. In the rat it is impossible selectively to transsect the OMN without also damaging sympathetic fibres that run alongside the sensory fibres in the OMN. The eyelids of the denervated right eye were sutured together to inhibit the development of a neuroparalytic keratitis. The animals were allowed to survive for 7 days. The efficiency of sensory denervation was demonstrated by the appearance of a total analgesy in the eye. Nerve fibres that completely disappeared following OMN transsection were considered sensory in nature. Nerve fibres that survived after the combined extirpation of SCG and transsection of the OMN were considered parasympathetic in nature.

Staining procedures

Only the eyes of the treated side were harvested in each rat of all experimental groups. Three different samples of eye tissues were harvested: (1) cornea, (2) iris and/or (3) choroid. For Bodian's staining (Bodian, 1939) the samples were fixed in a solution of 4% formaldehyde for 48 h, dehydrated, paraffin embedded and cut in serial transverse sections (10 µm). The sections were treated with 1% Protargol solution (colloidal silver), reducing solution (hydroquinone + sodium sulphite), 1% gold chloride solution, and 2% oxalic acid solution and counterstained with 0.03% aniline blue. All the nerve fibres and neurofibrils showed a black staining. All the sections were observed and photographed under a Zeiss photomicroscope (Karl Zeiss, Jena, Germany).

For demonstration of sympathetic nerve fibres, serial transverse fresh sections of the selected zones of the rat eyes (cornea, iris and choroid) of all experimental groups were cut on a cryostat. The method of Falck et al. (1962) and the glyoxylic acid-induced fluorescence technique as described by Qayyum & Fatani (1985) were used. Fresh sections were stained and immediately observed, analysed and photographed to prevent the diffusion and photo-decomposition of the fluorescence. The sections were examined and photographed under a Zeiss photomicroscope equipped with exciter and barrier filters and with a mercury lamp for observation of the fluorescence.

For demonstration of parasympathetic nerve fibres, serial transverse sections of the selected zones of the rat eyes (cornea, iris and choroid) of all experimental groups were fixed in a 10% formalin-containing 1% calcium chloride solution (1 min) and then processed according to the direct colouring thiocholine method (Karnowsky & Roots, 1964). Iso-octamethylpyro-phosphoramide (Iso-OMPA, Sigma) is able to differentiate intensity of staining of the various types of nerve fibres. Moreover, in our experiments, it was used as inhibitor of a specific cholinesterase (Du Bois et al. 1950).

Quantitative analysis of images

In order to provide quantitative values for each stained sample, a quantitative analysis of images (QAI) was performed on all the samples, in all the experimental conditions and after each type of staining, by means of a Leica Quantimet Analyser®. In all different types of staining and in all different experimental groups of rats only the same sections and/or the same regions of the eye were evaluated. QAI is able to synthesize in short tablular form a very much greater number of figures and/or photographs. The values reported in this paper represent the intensity of staining for each sample and are expressed in conventional units (CU) ± SEM. Further details on these units can be found in the Quantimet Manual (Microsystems Imaging Solutions, 1997).

Statistical analysis of the data

Preliminary analyses of each value obtiined by QAI were performed with the aid of basic sample statistics. Mean values, maximum and minimum limits, variations, standard error of the mean (SEM) and correlation coefficients were obtained via quantitative analysis of the photographs in the same experimental conditions, according to Serio (1986). Finally, a correlative analysis of the data was performed by comparing the significant differences for each group of samples with the corresponding values of the other homogeneous (i.e. in identical experimental conditions) samples.

Results

Identical points, for harvesting of the serial sections, were defined as homogeneous. Figure 1 shows images of three transverse sections of three different rat eyes as they appear at the operative microscope at a magnification of ×10. Line R is tangential to the extreme medial point of the right eye (treated) whereas line S is parallel to this and is 1 mm distant from the same line R. The harvested samples of the eye were: a, cornea; b, iris; and c, choroid.

Fig. 1.

Fig. 1

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Transverse sections of three different rat eyes observed by means of an operative microscope. Magnification ×10. The vertical line (S) indicates the point of harvesting of the samples of (a) the cornea, (b) the iris and (c) the choroid in serial sections. The vertical line (R) was tangential to the extreme left side of the eye bulb. The lines R and S are parallel and 1 mm apart. R, reference line; S, sample line.

Figure 2 shows a micrograph of choroids stained with Bodian's method before a minor parasympathectomy. Many nerve fibres show varicosities and beads along their length. On this photograph the QAI can be performed to quantify the number of nerve fibres present under normal conditions.

Fig. 2.

Fig. 2

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Transverse section of rat choroid before minor parasympathectomy and stained using Bodian's method for total nerve fibres (light microscopy; magnification ×600).

Figure 3 shows another (homogeneous) microscopic field after a minor parasympathectomy. We can observe a decrease in both the number and the area of the nerve fibres. From a comparison of the QAI values obtained before (Fig. 2) and after (Fig. 3) a partial parasympathectomy, we can calculate the number of the nerve fibres that have survived and disappeared.

Fig. 3.

Fig. 3

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Transverse section of rat choroid after minor parasympathetomy and stained using Bodian's method for total nerve fibres (light microscopy; magnification ×600).

A sample of cornea prior to surgical sympathectomy is shown in Figure 4. Many nerve fibres are located in the inner layers of the cornea. These catecholaminergic nerve fibres show beads and varicosities along their course.

Fig. 4.

Fig. 4

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Transverse section of rat cornea before sympathectomy and stained using the Falck and Hillap method for catecholaminergic nerve fibres and observed by means of fluorescence microscopy (magnification ×600). Numerous catecholaminergic nerve fibres are seen located in the cornea. The area occupied by these nerve fibres can be counted by QAI.

Figure 5 shows another microscopic field (homogeneous with Fig. 4) after surgical sympathectomy. Only a few remaining residual nerve fibres appear; the majority of the nerve fibres are granulated or have been destroyed.

Fig. 5.

Fig. 5

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Transverse section of rat cornea after sympathectomy and stained using the Falck and Hillarp method for catecholaminergic nerve fibres and observed by means of fluorescence microscopy. Only a few residual catecholaminergic nerve fibres are present in the cornea. The area occupied by these residual nerve fibres can be counted by QAI.

Owing to the numerous surgical and chemical procedures, different samples (cornea, iris and choroid) and different staining methods used, our quantitative results are summarized in three tables. Table 1 shows the values obtained by QAI before and after surgical and chemical sympathectomy. The iris and choroid are rich in catecholaminergic nerve fibres (about 65 CU), whereas the cornea is poor in catecholaminergic nerve fibres (about 15 CU). After sympathectomy, about 95% of the catecholaminergic nerve fibres are degenerated or have disappeared in all three studied structures of the eye (cornea, iris, choroid).

Table 1.

Values of catecholaminergic nerve fibres, stained using the Falck–Hillarp method and measured by QAI in three regions of the rat eye

Eye region Before sympathectomy n = 2 After surgical sympathectomy n = 8 After chemical sympathectomy n = 8
Comea 15.3 ± 2.9 1.6 ± 1.2 1.3 ± 0.4
Iris 64.4 ± 3.1 4.1 ± 0.8 0.9 ± 0.2
Choroid 63.1 ± 2.1 2.2 ± 0.6 0.6 ± 0.3
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The values reported in Table 1 are expressed in CU (see Methods), and are the residual catecholaminergic nerve fibres in each of the three structures of the eye. The values of Bodian staining were assumed as referee values.

Table 2 shows the values obtained by QAI before and after a minor and/or a major parasympathetomy. The iris and choroid possess a moderate quantity of Ache-positive nerve fibres (about 30 CU), whereas the cornea is notably lacking in Ache-positive nerve fibres (about 8 CU). After partial parasympathectomy, about 60% of the Ache-positive nerve fibres are degenerated or have disappered in all the three studied structures of the eye. After subtotal parasympathectomy, about 80% of the Ache-positive nerve fibres appear are degenerated or have disappeared

Table 2.

Values of Ache-positive nerve fibres, stained using the Karnowsky and Roots method and measured by QAI in three regions of the rat eye

Eye region Before parasympathectomy n = 2 After minor parasympathectomy n = 6 After major parasympathectomy n = 6
Comea 8.5 ± 1,2 4.8 ± 1.5 2.1 ± 0,6
Iris 30.8 ± 2.3 13.9 ± 1.2 3.4 ± 0,9
Choroid 31.6 ± 3.2 11.2 ± 1.9 4.3 ± 1.1
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The values reported in table 2 are expressed in C. U. (see methods) and are the residual Achepositive nerve fibres in each of the three structures of the eye. The values of Bodian staining were assumed as referee values.

Table 3 shows results for sensitive nerve fibres (or non-adrenergic, non-cholinergic nerve fibres) before and after the removal of the nervous ganglion. The cornea is highly sensitive whereas the iris and choroid are poorly sensitive. After removal of the sensitive ganglion, about 90% of nerve fibres are degenerated or have disappeared in all the studied structures of the rat eye.

Table 3.

Values of sensitive nerve fibres in three regions of the rat eye. These fibres were calculated as non-adrenergic, non-cholinergic nerve fibres

Eye region Before removal of the sensitive ganglion n=2 After removal of the sensitive ganglion n=6
Comea 78.2 ± 1.8 6.1 ± 1.3
Iris 3.4 ± 1.1 0.8 ± 0.6
Choroid 3.5 ± 0.6 0.6 ± 0.4
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The values reported in table 3 are expressed in C. U. (see methods) and are the residual sensitive nerve fibres in each of the three structures of the eye.

These sensitive nerve fibres were calculated as follows: Before = were considered as sensitive all the nerve fibres non adrenergic, non cholinergic (Total Bodian less values of table 1 and 2); After = the residual fibre of the same group after removal of the sensitive nervous ganglion. The values of Bodian methods were assumed as referee values.

Discussion

As reported above, numerous surgical and chemical procedures, using different eye samples and various staining methods were performed. The procedures were: (1) surgical sympathectomy, (2) chemical sympathectomy, (3) minor parasympathectomy, (4) major parasympathectomy and (5) removal of sensitive nervous ganglion. The staining methods were: (1) Bodian for total nerve fibres, (2) Falck–Hillarp and/or Qayyum and Fatani for catecholaminergic nerve fibres and (3) Karnowsky and Roots for Ache-positive nerve fibres. The samples of the eye were harvested from (1) cornea, (2) iris and (3) choroid.

Sham-operated rats served as controls. The values of this group are reported in our experimental data as ‘normal control’ and/or as ‘before procedure’. The total number of catecholaminergic nerve fibres that had degenerated and/or disappeared after sympathectomy were calculated by subtracting the values obtained after sympathectomy from the values obtained before sympathectomy. Three methods of staining were used and compared: those of (1) Bodian, (2) Falck–Hillarp and (3) Qayyum and Fatani.

The total number of cholinergic nerve fibres that had degenerated and/or disappeared after parasympathectomy was calculated by subtracting the values obtained after parasympathectomy from the values obtained before parasympathectomy. Two methods of staining were used and compared: (1) Bodian and (2) Karnowsky and Roots. Subtracting from the total nerve fibres the catecholaminergic plus the cholinergic fibres obtained the number of non-adrenergic, non-cholinergic nerve fibres. But if we consider this value before and after removal of the sensitive nervous ganglion we can obtain the number of sensitive nerve fibres.

The innervation of the eye is very complex. Our results confirm that the nerve fibres of the rat eye are sympathetic, parasympathetic and/or sensitive in nature. Falck-Hillarp, Ache and Bodian staining are probably not sufficient to discriminate the adrenergic, cholinergic and sensory nerves in human eyes. Moreover, these staining techniques can be applied only at the light microscopic and not at the electron microscopic level. For these reasons we studied the nerve fibres of the rat eye by means of a double staining technique (a general technique followed by a specific technique). The Bodian method, in fact, allows us to stain all the nerve fibres present in a sample. If we apply this method in two homogeneous samples before and after a denervation we are able to count by QAI the number of fibres that have survived or have degenerated. Moreover, if we compare the data for the general stain before and after the denervation with the values obtained with a specific stain (Ache or Falck–Hillarp) in the homogeneous samples before and after the denervation we are able to obtain useful data regarding the innervation of the eye, which has proved a complex area of research (Ruskell, 1993, 2003, 2004; van der Werf et al. 1996; Toth et al. 1999). Our QAI data, obatined before and after denervation of the rat eye, represent only ‘comparative’ values.

Therefore, it is impossible to describe a precise map of the innervation of the various regions of the eye. In fact, all the regions of the eye show an overlapping of adrenergic, cholinergic and/or sensitive nerve fibres. Only the experiments of selective denervation allow us to distinguish between the various types of nerve fibres.

The intraocular distribution of sympathetic and parasympathetic nerve fibres has been well studied, but little is known regarding sensitive nerve supply.

Our results confirm that, in the rat eye, sensory nerve fibres from the ophthalmic division of the trigeminal nerve enter the ciliary processes and supply all the tissues of the anterior segment. The sensory nerves are diffusely distributed and terminate as unmyelinated fibres without special endings.

In conclusion, experimental sectioning of different types of ocular nerve fibres and/or removal of the peripheral nerve ganglia is useful for establishing the nature and source of each of these nerve fibre types. Moreover, it is possible to observe which neurotransmitter disappears after removal of the different types of the ocular nervous ganglia.

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