Morphometric Analysis of the Optic Nerve in Experimental Hydrocephalus-Induced Rats

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Original Paper

Pediatr Neurosurg 2011;47:342–348 DOI: 10.1159/000337728

Morphometric Analysis of the Optic Nerve in ExperimentalHydrocephalus-Induced Rats

Betina Aisengart a João Kazuyuki Kajiwara b Karinna Veríssimo Meira a

Luiza da Silva Lopes a

Departments of a Surgery and Anatomy, and b Cellular and Molecular Biology and Pathogenic Bioagents, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto , Brazil

Introduction

Obstructive hydrocephalus is a syndrome caused by conditions that obstruct the cerebrospinal fluid flow in the brain ventricles [1] . It is characterized by significant changes in the water content and transport in the brain [1] . These changes are always accompanied by brain de-formation [1–4] . A clinical study using ultrasound tech-nique observed a significant correlation of values for optic nerve sheath diameter in a group of shunted hydrocephal-ic patients with shunt malfunction compared with a con-trol population [5] . Hydrocephalus unrelated to tumor and hereditary/genetic diseases is the third most fre-quently observed cause of optic atrophy, and recurrent el-evation of intracranial pressure (ICP) can have serious vi-sual consequences for a child [6–8] . The aim of the present study was to evaluate morphometric alterations in pup rat optic nerves submitted to experimental hydrocephalus.

Material and Methods

All animals were treated in accordance with the Colégio Brasileiro de Experimentação Animal guideline, and protocols were approved by the local institutional animal ethics committee. Seventy-eight 7-day-old Wistar rats were used without sexual dis-

Key Words

Experimental hydrocephalus � Optic nerve

Abstract

Purpose: It was the aim of this study to investigate changes caused to the optic nerve of rats submitted to experimental hydrocephalus through morphometric analysis. Method: At postnatal day 7, the rats underwent injection of kaolin into the cisterna magna, were sacrificed at postnatal day 14, 21 or 28, and the right optic nerves were dissected. We analyzed the area, minor diameter, densities of oligodendrocytes and astrocytes, total and damaged fibers as well as the relation-ship between damaged and total fibers. Results: At postna-tal day 14, there was a reduction in the density of astrocytes and damaged fibers when compared to the controls. At postnatal day 21, the area and the minor diameter were re-duced compared to the controls, and the densities of oligo-dendrocytes and damaged fibers were increased compared to the controls. At postnatal day 28, there was a reduction in the area and the minor diameter and an increase in the den-sities of oligodendrocytes, astrocytes and damaged fibers when compared to controls. Conclusion: The optic nerve of rats submitted to experimental hydrocephalus suffers mor-phometric changes. Copyright © 2012 S. Karger AG, Basel

Received: May 5, 2011 Accepted after revision: March 2, 2012 Published online: May 9, 2012

Betina Aisengart Federal University of Parana, Centro Politécnico – Biology Sciences Sector Department of Physiology, Laboratory of Pain – room 116 PO Box 19031, Jardim das Américas, Curitiba, PR 81530-130 (Brazil) Tel. +55 41 3361 1714, E-Mail betina77 @ gmail.com

© 2012 S. Karger AG, Basel1016–2291/11/0475–0342$38.00/0

Accessible online at:www.karger.com/pne

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http://dx.doi.org/10.1159%2F000337728

Morphometric Analysis of the Optic Nerve in Rats

Pediatr Neurosurg 2011;47:342–348 343

tinction. Each litter consisted of the mother rat and 8–10 newborn rats. Each pup rat was held by an auxiliary, who held the head with one hand and the body with the other, bending the animal’s neck, leaving the posterior cervical area free. By palpation, the space be-tween the posterior extremity of the foramen magnum, on the oc-cipital bone, and the posterior arch of the first cervical vertebra was identified. A Mise 0.3 odontologic needle, equipped with a short bevel, was used for the suboccipital percutaneous injection of 0.4 ml of a kaolin suspension (Merck � ) diluted in distilled water of 20%. One rat of each litter was not injected and used as a control. Following this, the rats were put back into their cages with their mothers. All the injected animals were observed daily to see if there were any clinical or behavior alterations. Those rats which did not develop hydrocephalus after the injection were dismissed and were not included in the number of animals of each group.

The animals at postnatal day 14 (n = 26), 21 (n = 26) or 28 (n = 26; P14, P21 and P28 group, respectively) were anesthetized with a ketamine and xylazine mixture, submitted to transcardiac

perfusion with phosphate-buffered saline solution (about 1 ml/g of the animal’s weight) and fixed with 4% paraformaldehyde and 2% glutaraldehyde mixture in 0.1 M , pH 7.3, phosphate buffer. The rats were decapitated, their brains removed in block by a craniec-tomy of the vertex and immersed in the same fixative solution of formaldehyde and glutaraldehyde for 1 24 h at 4 ° C. After washing with 0.1 M phosphate buffer, the brains were sectioned at the me-dium sagittal plane, labeled in 2 groups of severe and mild hydro-cephalus, and the right optic nerve was dissected. The samples obtained of the optic nerve were fixed in a 1% osmium tetroxide in 0.2 M phosphate buffer for 2 h, at 4 ° C, washed with 0.1 M phos-phate buffer and then dehydrated in an increasing series of ace-tone and embedded in Araldite � . The inclusion was done to per-mit that the optic nerve fibers could always be sectioned transver-sally. After polymerization of the resin, the blocks were cut into 0.5-mm-thick sections and stained with 1% toluidine blue.

The sections were analyzed under a light microscope and pho-tographed through the Axioskop 2 Plus with a AxioCam HRC

Table 1. G roup P14 (n = 26)

Area, mm2 Minor diam-eter, mm

Oligodendro-cytes/mm2

Astrocytes/mm2

Totalfibers/mm2

Damagedfibers/mm2

Relationdamaged/total fibers

Control (n = 5) 639,105 713,060 2,363 2,511 96,840 2,663 0.45Severe (n = 10) 627,391 688,935 2,493 2,154 58,710 26,685 0.51Mild (n = 11) 684,130 754,111 2,367 2,113 71,945 33,598 0.52




Astrocytes/mm2

Totalfibers/mm2

Damagedfibers/mm2

Relationdamaged/total fibers

Control (n = 5) 414,841 614,927 1,330 1,933 151,240 25,220 0.17Severe (n = 13) 219,098 418,637 1,663 1,996 133,292 38,653 0.29Mild (n = 8) 240,129 472,572 1,923 2,110 120,213 45,096 0.39




Astrocytes/mm2

Totalfibers/mm2

Damagedfibers/mm2

Relationdamaged/totalfibers

Control (n = 5) 639,105 713,060 2,363 2,511 96,840 2,663 0.45 Severe (n = 8) 627,391 688,935 2,493 2,154 58,710 26,685 0.51Mild (n = 13) 684,130 754,111 2,367 2,113 71,945 33,598 0.52

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Aisengart /Kajiwara /Veríssimo Meira /da Silva Lopes

Pediatr Neurosurg 2011;47:342–348344

camera. We analyzed, through six randomized fields with a total area of 10,000 � m 2 (0.01 mm2), the total myelin fibers, the dam-aged myelin fibers, astrocyte and oligodendrocyte densities; the area and minor diameter were collected by the software image J 1.38x version available for free by the National Institutes of Health, USA. Statistical analysis was performed using the PROC MIXED procedure of SAS � 8.0 software, and the data were analyzed by mixed-effects linear model (random effects and fixed). p ̂ 0.05 was considered statistically significant.

Results

Area and Minor Diameter The P14 group ( table 1 ) showed no statistically signifi-

cant difference in the area and minor diameter, but the P21 group ( table 2 ) showed a statistically significant re-

duction in the area and minor diameter between the con-trol and severe hydrocephalus animals; we also observed a statistically significant reduction in the minor diameter between control and mild hydrocephalus animals ( fig. 1 ). In the P28 group ( table 3 ), we found a statistically signif-icant reduction in the area and minor diameter between the control and severe hydrocephalus as well as between the mild and severe hydrocephalus rats ( fig. 1 ).

Oligodendrocyte Density The P14 group ( table 1 ) showed no statistically signifi-

cant difference in the density of oligodendrocytes, but a statistically significant increase between the control and mild hydrocephalus animals in the P21 group ( table 2 ) and between the control and severe hydrocephalus ani-mals in the P28 group ( table 3 ) was observed ( fig. 1 ).

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Fig. 1. Bar chart showing the mean 8 SD area ( a ), minor diameter ( b ), as well as the densities of oligodendrocytes ( c ) and astrocytes ( d ) of the P14, P21 and P28 groups. It appears that the area de-creases in the P21 and P28 groups when compared to the P14 group, both in control and in hydrocephalus animals; the minor diameter decreases in the P21 group and increases in the P28 group when compared to the P14 group, both in control and in

hydrocephalus animals. There is a reduction in the density of oli-godendrocytes among the three groups that remained constant between postnatal days 21 and 28, both in control and hydroceph-alus animals. There is a reduction in the density of astrocytes among the control animals but this does not occur in hydroceph-alus rats whose densities remain constant.

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Astrocyte Density The astrocyte density in the P14 group ( table 1 ) showed

a statistically significant reduction between the control and mild hydrocephalus rats; between the control and severe hydrocephalus rats in the P21 group ( table 2 ), there was no statistically significant difference. However, the P28 group ( table 3 ) showed an increase in astrocyte den-sity between the control and mild hydrocephalus animals ( fig. 1 ).

Fiber Density There was no statistically significant difference be-

tween groups when comparing the density of total nerve fibers. The damaged nerve fiber density (P14 group) was statistically significantly reduced between the control and severe hydrocephalus animals ( table 1 ), whereas the P21 ( table 2 ) and P28 ( table 3 ) groups showed an increase among controls and severe/mild hydrocephalus animals. The relation of damaged nerve fibers/total nerve fibers in the P21 group showed a statistically significant in-crease between the control and mild hydrocephalus rats

( table 2 ). The P28 group ( table 3 ) showed an increase among the control and severe/mild hydrocephalus rats ( fig. 2 ).

Histology Observation of the optic nerve sections stained with

toluidine blue showed that in relation to the 14-day-old rats, the myelinated fibers of the control ( fig. 3 a) and mild ( fig. 3 c) hydrocephalic animals are dispersed, i.e. they present a lower number than the severe ( fig. 3 b) hy-drocephalus rats. Neuroglial cells are present in a great number with big nuclei and evident nucleoli ( fig. 3 a–c). In relation to the 21-day-old rats, the number of myelin-ated fibers is increased although compacted. The neuro-glial cells are present in a reduced number in relation to the 14-day-old animals ( fig. 3 d–f). Compared to the 28-day-old rats, the controls ( fig. 3 g) present myelinated fibers which are even more exuberant in number and density. These fibers are bigger and better defined ( fig. 3 g–i).

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Fig. 2. Bar chart showing the mean 8 SD densities of damaged myelinated fibers ( a ), total myelinated fibers ( b ) and the relation of damaged/total myelinated fibers ( c ) of the P14, P21 and P28 groups. It appears that there is a great increase in the density of total myelinated fibers among the three groups that remained constant between postnatal day 21 and 28. Further, there is a re-duction in the density of damaged myelinated fibers in the P21 group and an increase in the P28 group, but this does not occur in hydrocephalus rats whose densities increase progressively. It appears that there is a reduction in the relation between damaged and total myelinated fibers in the P21 group and an increase in the P28 group, both in control and severe hydrocephalus animals, but not in mild hydrocephalus rats whose relation increases pro-gressively.

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a b c

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Fig. 3. a–c Photomicrographs of the optic nerve of control ( a ), se-vere ( b ) and mild ( c ) hydrocephalus rats at postnatal day 14. There are few and disorganized myelinated fibers and abundant inter-stitial space; neuroglial cells are present in great number. d–f Pho-tomicrographs of the optic nerve of control ( d ), severe ( e ) and mild ( f ) hydrocephalus rats at postnatal day 21. The interstitial space is not as abundant and there is a great amount of myelin-

ated fibers. In control and mild hydrocephalus rats, the fibers are in a bundle organization and the number of neuroglial cells is re-duced. g–i Photomicrographs of the optic nerve of control ( g ), severe ( h ) and mild ( i ) hydrocephalus rats at postnatal day 28. In-terstitial space is almost nonexistent and the amount of myelin-ated fibers is very large and organized in a bundle. Toluidine blue. ! 100.

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head injury patients, found that of the 59 patients with suspected intracranial injury and possible elevated ICP, 8 patients with an ONS diameter 6 5.00 mm had computed tomographic findings that correlated with elevated ICP.

Non-invasive technique is well tolerated in children and may have a predictive role in the assessment of hy-drocephalus or shunt malfunction when brain imaging reveals static ventricular configuration [11] . The fact that the ONS diameter had a reasonable sensitivity for any traumatic head injury suggests the need for further re-search in other settings and age ranges for the evaluation, stratification and possible acute treatment of head injury [12] .

We suggest that the ONS diameter offers further tools for physicians who treat head injuries beyond physical examination and that multiple examinations are neces-sary to evaluate the ONS diameter pattern for each pa-tient to determine the clinical relevance in the individual.

We found a reduction in the density of oligodendro-cytes of control rats at postnatal days 14–21, and thereaf-ter, a slight decrease in order to maintain a constant amount. This pattern was also found in mild hydroceph-alus animals; however, in severe hydrocephalus rats, there was a slight increase between days 21 and 28. This may happen as a failed attempt of the body in reversing the process of demyelination of the optic nerve. In 2008, De Maman et al. [13] , studying early iron deficiency, found that the number of oligodendrocytes of the anemic group was reduced in comparison with the control group, the oligodendrocyte density was increased in the recovery group and the number of these cells remained inferior in the anemic and recovery groups in comparison with the control group.

In our study, we found that the density of astrocytes is reduced both in the severe and mild hydrocephalus ani-mals, compared to the control rats, and this reduction is more significant at postnatal day 14. In 2009, Da Silva Lopes et al. [14] found that in control mice immunohis-tochemical detection of glial fibrillary acidic protein re-vealed astrocytes with delicate processes around blood vessels in white matter locations and the subventricular zone at the angle of the ventricle.

We observed that the density of fibers was growing. The rat is born with a small amount, possibly explained by the fact that the fibers are not completely myelinated until postnatal day 14; at postnatal day 21, a significant increase in the density of fibers is presented which re-mains constant until postnatal day 28. The hydrocephal-ic animals follow the same behavior but there is a de-crease in the density of fibers when compared to age-

Discussion

This work highlights the importance of routine oph-thalmologic examination in patients with hydrocepha-lus. Among the sequelae of persistent raised ICP are oph-thalmologic signs and symptoms, including cranial nerve palsies, visual field deficits, papilledema and vision loss [9] .

According to our studies, the area and the minor di-ameter of the optic nerve of control rats has been low at postnatal day 21 and has increased statistically after day 28 when compared with 14-day-old animals. This devel-opment is also seen with the number of cells inside the nerve, possibly because the brain at postnatal day 14 has abundant interstitial space and the brain at day 21 has more cells than interstitial space which, however, are not organized and fully myelinated. At postnatal day 28, the brain has so many fully myelinated cells, it has lost inter-stitial space, as seen in figure 3 . When we compared the area and the minor diameter of control rats with hydro-cephalic animals, we found that there is a significant re-duction in the minor diameters when the rats showed se-vere hydrocephalus and a slight reduction when the de-gree of hydrocephalus was mild, and that by comparing the grades of mild and severe hydrocephalus, time influ-enced the size of the area and the minor diameter. In 2008, Watanabe et al. [10] by investigating the potential use of optic nerve sheath (ONS) diameter as a possible marker of ICP found that the normal ONS diameters 1 4 mm posterior to the globe are 5.52 8 1.11 mm in ma-jor diameter and 5.2 8 0.9 mm in minor diameter with a subdural pressure of 9–30 cm H 2 O (mean 19 8 8.2)before surgery. The diameters of the ONS decreased to 3–5.7 mm (mean 4.8 8 0.9) after full recovery from symptoms, and thus, the authors concluded that an in-creased ONS diameter measured on coronal orbital im-ages is a strong indicator of increased ICP and helps to differentiate between passive subdural fluid collection due to brain atrophy and subdural hygroma with in-creased ICP [10] .

In 2009, McAuley et al. [11] , while measuring the sheath diameter using transorbital ultrasound as a clini-cal assessment indicator of developing hydrocephalus in the pediatric population, found that the ONS diameter is increased in symptomatic patients from their own base-line values which may have a predictive role in the assess-ment of hydrocephalus or shunt malfunction when brain imaging reveals static ventricular configuration. In 2007, Tayal et al. [12] , using sonographic measurement of the ONS diameter to detect findings of increased ICP in adult

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matching controls, which may be due to the delay in myelination of the fibers being more significant in the severe degree of hydrocephalus.

In 2003, Da Silva Lopes et al. [15] found that hydroce-phalic animals at postnatal day 14 presented a smaller number of myelinated fibers of the corpus callosum when compared to their controls; however, the hydrocephalic rats at postnatal day 21 showed a higher number of my-elinated fibers and more densely compacted than the controls, and the animals at postnatal day 28 also showed an important loss of myelinated fibers which were dis-persed, with small areas of compaction.

In our study, we conclude that the progression time of hydrocephalus may be the most important factor, rather than the degree of ventricular dilation, in the injury of the optic nerves in young rats because the tissue subjected to prolonged ischemia suffers deleterious effects and often leads to injury and cell death by necrosis and apoptosis. Ischemia leads to various degrees of ocular visual loss due to irreversible loss of neuronal tissue [16] . In 70% of indi-viduals with demyelinating diseases, the optical system is affected. Eventually, the ability to remyelinate the optic nerve is low and results in permanent visual loss [17] . With the increased longevity of patients with hydroceph-alus, issues such as quality of life become important, and

eye problems include these issues. To ensure an early di-agnosis and treatment of hydrocephalus, both pediatric ophthalmologists and neurosurgeons should be better informed about ophthalmologic complications of hydro-cephalus. A pediatric ophthalmologist should undertake regular examinations of visual acuity and field such as regular ophthalmic supervision to maintain the best pos-sible standard of vision in children with hydrocephalus. With such teamwork, the earliest ophthalmic signs of in-creased ICP can be detected in patients where the diag-nosis is in doubt.

In conclusion, the optic nerve of rats with experimen-tal hydrocephalus undergoes morphometric changes. The degree of ventricular enlargement influences the ex-tent of the area and the minor diameter. Experimental hydrocephalus generates a delay in the myelination of nerve fibers due to a reduction in the density of oligoden-drocytes. The exposure time of the optic nerve to hydro-cephalus may be the most important factor producing in-jury, rather than the degree of ventricular dilation.

Acknowledgment

We thank Mrs. Izilda Rodrigues for her excellent technical as-sistance. This work was supported by CNPq (Brazil).

References

1 Kaczmareck M, Subramaniam RP, Neff SR: The hydromechanics of hydrocephalus: steady-state solutions for cylindrical geom-etry. Bull Mat Biol 1997; 59: 295–323.

2 Del Bigio MR, Bruni E, Fewer HD: Human neonatal hydrocephalus. An electron micro-scopic study of the periventricular tissue. J Neurosurg 1985; 63: 56–63.

3 Kiefer M, Eymann R, Von Tiling S, Müller A, Steudel W-I, Booz K-H: The ependyma in chronic hydrocephalus. Childs Nerv Syst 1998; 14: 263–270.

4 Del Bigio MR, McAllister JP II: Hydrocepha-lus – pathology; in Choux M, Di Rocco C, Hockley AD, Walker ML (eds): Pediatric Neurosurgery. London, Churchill Living-stone, 1999, pp 217–236.

5 Newmann WD, Hollman AS, Dutton GN, Carachi R: Measurement of optic nerve sheath diameter by ultrasound: a means of detecting acute raised intracranial pressure in hydrocephalus. Br J Ophthalmol 2002; 86: 1109–1113.

6 Mudgil AV, Repka MX: Childhood optic at-rophy. Clin Exp Ophthalmol 2000; 28: 34–37.

7 Molia L, Winterkorn JMS, Schneider SJ: Hemianopic visual field defects in children with intracranial shuts: report of two cases. Neurosurgery 1996; 39: 599–603.

8 Rudolph D, Sterker I, Graefe G, et al: Visual field constriction in children with shunt-treated hydrocephalus. J Neurosurg Pediatr 2010; 6: 481–485.

9 Carruth BP, Bersani TA, Hurley PE, Ko MV: Visual improvement after optic nerve sheath decompression in a case of congenital hydro-cephalus and persistent visual loss despite intracranial pressure correction via shunt-ing. Ophthal Plast Reconstr Surg 2010; 26: 297–298.

10 Watanabe A, Kinouchi H, Horikoshi T, Uchida M, Ishigam K: Effect of intracranial pressure on the diameter of the optic nerve sheath. J Neurosurg 2008; 109: 255–258.

11 McAuley D, Paterson A, Sweeney L: Optic nerve sheath ultrasound in the assessment of paediatric hydrocephalus. Childs Nerv Syst 2009; 25: 87–90.

12 Tayal VS, Neulander M, Norton HJ, Foster T, Saunders T, Blaivas M: Emergency depart-ment sonographic measurement of optic nerve sheath diameter to detect findings of increased intracranial pressure in adult head injury patients. Ann Emerg Med 2007; 49: 508–514.

13 DeMaman AS, Homem JM, Lachat J-J: Early iron deficiency produces persistent damage to visual tracts in Wistar rats. Nutr Neurosci 2008; 11: 1–7.

14 Da Silva Lopes L, Slobodian I, Del Bigio MR: Characterization of juvenile and young adult mice following induction of hydrocephalus with kaolin. Exp Neurol 2009; 219: 187–196.

15 Da Silva Lopes L, Machado HR, Lachat J-J: Study of corpus callosum in experimental hydrocephalic Wistar rats. Acta Cir Bras 2003; 18: 10–14.

16 Obolensky A, Berenshtein E, Konijn AM, Banin E, Chevion M: Ischemic precondi-tioning of the rat retina: protective role of ferritin. Free Radic Biol Med 2008; 44: 1286–1294.

17 Guazzo EP: A technique for producing de-myelination of the rat optic nerves. J Clin Neurosci 2005; 12: 54–58.

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Morphometric Analysis of the Optic Nerve in Experimental Hydrocephalus-Induced Rats

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