Morphometric analysis of the fascicular organisation of the optic nerve Morfometrijska analiza fascikularne organizacije opti kog nerva

Background/Aim. The optic nerve is anatomically observed in four segments: intrabulbar, orbital, canalicular, and cranial. According to the literature, the surface of the transversal cut of the nerve is different through it. The aim of this study was to evaluate the fascicular organisation of the optic nerve, throughout its three segments from the eye. Methods. Five pairs of optic nerves, obtained from the autopsies were examined. Using Heidenhain's (azan) staining, the cuts were prepared for microscopy. Morphometric analysis was performed using the stereological methods for morphometric cytology – the Weible’s testing system M42. The following measures were established: the surface of the transverse cut of the nerve, the entire surface of fasciculi, the entire surface of connective tissue and blood vessels, the number of fasciculi, the surface of a single fasciculus. Results. The surface of the transverse cut of the nerve was found to grow from the orbital to the cranial segment, as well as the entire surface of fasciculi. While their number is significantly lower in the cranial segment, the number of fasciculi varied slightly between the orbital and the canalicular segment. The surface of a single fasciculus grows from the bulb to the chiasma. There is probable a cause to believe that this may be due to fusion of the “small” fasciculi in the orbitocranial direction. Conclusion. There are significant differences among the examined parameters of the different parts of the optic nerve.


Introduction
While the anatomo-topographical, neuro-ophthalmologi cal, and neurosurgical aspects of the optic nerve have been well established [1][2][3] , its ultrastructure has yet to be defined outside of animal models 4 .
The embryological origin of the retina and the optic nerve is neuroectoderm in nature, closely linked to the de-velopment of the central nervous system 5 .The axons of the retinal ganglion cells pass through the outer retinal layers, the choroid layer, and the lamina cribrosa, where they obtain a myelin layer and form the fasciculi of the optic nerve 6 .Since the axons of the optic nerve have a central origin, oligodendrocytes are involved in forming its myelin layer, whilst the fasciculi are formed by astrocytes and connective tissue 4 .
The optic nerve is anatomically observed in four segments: intrabulbar, orbital, canalicular, and the cranial 7 .According to the outcomes of Jonas et al. 8 study, the surface of the transversal cut of the nerve is different throughout, ranging from 3.93 mm 2 to 8.15 mm 2 (mean 5.92 ± 1.06 mm 2 ), with 73.2 ± 5.8% of the surface belonging to fasciculi (optical fibres).
The aim of this study was to evaluate the fascicular organisation of the optic nerve, throughout its three segments from the eye.

Methods
We examined five pairs of optic nerves, obtained from the autopsies of 3 male and 2 female bodies, of 42 to 74 years of age, who showed no signs of central nervous pathology.The post-mortem interval (the time from death to autopsy) ranged from 10 to 24 hours in length.
Removal of the brain was done according to the standard autopsy techniques, cutting the optic nerves in the region of the optic chiasma.After removing the orbital roof and opening the optic channel, a detailed preparation of the optic nerves was performed.The orbital, canalicular, and cranial segments of the nerve were all separated.
Using Heidenhain's (azan) staining 9 , the cuts were prepared for microscopy.Morphometric analysis was performed by the stereological methods for morphometric cytology; specifically by the Weible's testing system M42, with 42 test dots and 21 test lines 10,11 .
The following measures were established: the surface (area) of the transverse cut of the nerve (A N ) and its mean value ( A N ), the entire surface (area) of fasciculi (A F ) and their mean value ( A F ); the entire surface (area) of connective tissue and blood vessels (A CV = A N -A F ), and their mean value ( A CV ); the number of fasciculi (N); the surface (area) of a single fasciculus (A FS ) and its mean value ( A FS ) -[A N (or A F , or A CV , or A FS ,) = P D • a] were P D = the number of dots of the test system that are present on the examined surface; a = the area that belongs to a single dot of the testing system calculated by the formula a = (d 2 • 3) 2; and d = the distance between two dots, depending on microscope magnification (for magnification 10, d = 0.16 mm, a = 0.0221702 mm 2 ).The number of fasciculi was established by manual counting from the microphotographs of a magnification 25 to 75 (Figure 1).The standard statistical protocol for descriptive statistics was used, including Student's t-test for independent samples at 95% confidence intervals.

Results
Regarding sex and age variations between the examined parameters of the nerves no differences were found (sex: p < 0.01; age: p = 0.03), so the sample was homogenous.
The values of the measured morphometric parameters (A N , A F , A CV , and A FS ) were different for the three segments of the nerve (Tables 1 and 2).
By using t-test, no statistically significant difference was found between the orbital and the canalicular surface of  The values are the mean value ± standard deviation and standard error in parentheses.
the nerve, (p = 0.313), as well as between the canalicular and the cranial surface of the nerve (p = 0.053).Nonetheless, there was a statistically significant difference between the orbital and the cranial segment (p = 0.016).By analysing the A F , a statistically significant difference was found between the orbital and the cranial (p = 0.005), as well as between the canalicular and the cranial segment (p = 0.021), but not between orbital and canalicular one (p = 0.131).
No significance was found between the A CV of any segment of the nerve.
Table 2 presents the number of fasciculi by the segments.According to t-test, a high statistically significant difference was found between the canalicular and the cranial, as well as between the orbital and the cranial (p < 0.000), but not between the orbital and the canalicular segment (p = 0.415).In addition, Table 2 notes the values of A FS .A statistically significant difference was found among all the three segments: orbital vs canalicular (p = 0.013) canalicular vs cranial (p < 0.000), orbital vs cranial (p < 0.000).
Figure 2 shows the ratios between the A F and the A CV for each segment of the nerve.

Discussion
We used stereological analysis simple and cheap, but somewhat out-of-fashion method to establish the morphometric parameters of histo-anatomy.The method is especially useful for cases in whom qualitative analysis does not prove sufficient, e.g. for small differences between the groups or for a high variability in parameters which hides the differences between them.Moreover, it is a well-established fact that the interobserver difference is recorded as extant in similar cases 12 .Both of these factors contributed to choosing of the morphometric method of the Weible's testing system M42 for stereological examination.
The results of the study demonstrate significant differences between the examined parameters of the different parts of the nerve.Therein, the surface of the whole nerve is significantly different between the orbital and the cranial segment, which is in accordance with the outcomes of Jonas et al. study 8 .Since the surfaces under study here were examined in the segment-by-segment fashion, the presented results are more valuable in gaining a better understanding of the fascicular organisation of the nerve.
The surface of the transverse cut of the nerve was found to grow from the orbital to the cranial segment, as well as the entire surface of the fasciculi.Similar results were presented in Tao et al. study 13 .While their number was significantly lower in the canalicular segment, the number of the fasciculi varied slightly between the orbital and the canalicular segment.The surface of a single fasciculus grows from the bulb to the chiasma.A similar finding has been repeatedly published for animal models 14 -17 .Jeffery et al. 18 also noticed the lowest number of fasciculi to be in the central area of the orbital segment, the highest pic in the canalicular segment, and a constant decrease throughout the cranial segment.
Even numerous studies demonstated the age-related decrease in fibers in the optic nerve, with cca 5,000 fibers per year [19][20][21] , the paper under review here did not establish the decrease in the number of fasciculi, nor the surface of a single fasciculus regarding age.
The fascicular organisation of the optic nerve is strongly dependent on the number of fibrous astrocytes, namely the axon's growth is dependent on the number of astrocytes and their processes.A lower amount of connective tissue in the cranial segment is responsible for the wider spread of optical fasciculi 16,17,19,[21][22][23] .A possible link between the fascicular reorganisation and the lower glial volume in the cranial segment may stem from the recomposition of optic fibres in the chiasma.Taking the A FS and N F in the cranial segment into consideration, a similar hypothesis may also be posited of that a significantly higher A FS and lower N F in the cranial segment may be the consequence of the lower number of fibrous astrocytes and the wider spread of the optic fibres.In addition, due to the fusion of the "small" fasciculi, their number decreases in the cranial segment 4,18 .The final results suggest that the fusion of the "small" fasciculi begins from the orbital segment (N F(orbital) > N F(canalicular); A KS(orbital) > A KS(canalicular) ).In addition, fascicular and glial organisation in related nerve segements are followed with capilary rearrangement 24 .
Having in mind the established fascicular nerve organisation, especially fascicular fusion, pathology and trauma of different segments would differ.Smaller injuries (fracture of the surrounding bones, or nerve contusion), or even lighter nerve compression (oedema and varoius types of haemorrhages) in cranial direction of the nerve can damage smaller number of fasciculi but with higher negative impact on eye sight.

Conclusion
The transversal surface of the optic nerve, as well as the entire fascicular surface grows from the orbital to the cranial segment.Conversely, the surface of the connective tissue and blood vessels decreases upon reaching the chiasma, but not significantly.
The number of fasciculi decreases from the orbital to the cranial segment, while the surface of a single fasciculus increases in the same direction.There is probable a cause to believe that this may be due to fusion of the "small" fasciculi in the orbitocranial direction.