Optic Nerve Sheath Fenestration With aNovel Wavelength Produced by the Free

Electron Laser (FEL)Karen M. Joos, MD, PhD,1* Jin H. Shen, PhD,1 Debra J. Shetlar, MD, PhD,1–3 and

Vivien A. Casagrande, PhD1,4

1Department of Ophthalmology and Visual Sciences, Vanderbilt University, Nashville, Tennessee2Department of Pathology, Vanderbilt University, Nashville, Tennessee

3Cullen Eye Institute, Baylor College of Medicine, Houston, Texas4Departments of Cell Biology and Psychology, Vanderbilt University, Nashville, Tennessee

Background and Objective: To determine whether 6.45-mm free electronlaser (FEL) energy can successfully perform optic nerve sheath fenes-tration and to compare the acute and chronic cellular responses withthis surgery.Study Design/Materials and Methods: Optic nerve sheath fenestrationwas performed in rabbits by using either FEL energy (< 2.5 mJ, 10 Hz,325-mm spot size) or a knife. The optic nerve integrity and glial re-sponse were evaluated histologically acutely or 1 month postopera-tively.Results: The FEL at low energy effectively cut the optic nerve sheathswith minimal reaction in the underlying nerve. With FEL or knife sur-gical techniques, a mild astrocytic hypertrophy only adjacent to thefenestration was observed acutely in the glial fibrillary acidic protein(GFAP) -immunoreacted sections. The chronic healing responses aftereither technique appeared similar with: (1) a thin fibrous scar at thefenestration site, (2) cells uniformly distributed (hematoxylin and eo-sin), and (3) up-regulation of GFAP and S100b in astrocytes adjacent tothe fenestration site.Conclusion: The FEL at low energy performs an optic nerve sheathfenestration in a small space with ease. Both FEL and knife incisionscause a similar rapid glial response near the fenestration site that re-mains 1 month later. Lasers Surg. Med. 27:191–205, 2000.© 2000 Wiley-Liss, Inc.

Key words: glial cell; immunocytochemistry; laser; optic nerve; optic nerve sheathfenestration; surgery; papilledema

INTRODUCTION

Pseudotumor cerebri is characterized byheadaches, visual obscurations, marked visionloss, deterioration of the visual field, and opticdisc swelling. In adults, the course can be moreprotracted with associations including obesity,middle ear disease, pregnancy, corticosteroid useor withdrawal, high-dose vitamin A, nalidixicacid, tetracycline, nutrition disorders, toxins, im-mune disorders, or after mild head trauma [1,2].Increased intracranial pressure also can occursecondary to cerebral venous outflow obstruction[3,4]. The increased pressure causes headachesand is transmitted along the subarachnoid space

of the optic nerve to produce papilledema of theoptic disc [5]. Treatment is aimed at decreasingthe intracranial pressure around the optic nerves.

Contract grant sponsor: Department of Defense Medical FreeElectron Laser Program, Office of Naval Research; Contractgrant number: N00014-94-1-1023; Contract grant sponsor:Research to Prevent Blindness, Inc.; Contract grant sponsor:National Institutes of Health; Contract grant number: CoreGrant EY08126.

*Correspondence to: Karen Joos, MD, PhD, Department ofOphthalmology and Visual Sciences, 1215 21st AvenueSouth, 8017 MCE, Nashville, TN 37232-8808.E-mail: karen.joos@mcmail.vanderbilt.edu

Accepted 17 March 2000

Lasers in Surgery and Medicine 27:191–205 (2000)

© 2000 Wiley-Liss, Inc.

Surgical treatments are performed when medicaltherapies fail to prevent vision loss or are not tol-erated by the patient [6].

Optic nerve sheath decompression has beensuccessful in preventing and even restoring lostvision [7,8]. However, it can be technically diffi-cult to expose and then cut a window or incisemultiple slits typically with a knife or scissors inthe sheath without disturbing the underlying op-tic nerve. Also, approximately 30% of the success-ful procedures may fail within 5 years with visualdeterioration [8]. If pseudotumor cerebri is notsuccessfully treated, gliosis of the optic nervehead may cause permanent severe vision loss orvisual field loss [7]. Even with treatment, partialloss of vision may occur in some patients. Histo-logic loss of parvocellular and magnocellular cellsin the lateral geniculate nucleus has been ob-served in pathologic specimens [9].

Improvements in the surgical techniquehave been investigated, including approachingthe optic nerve medially instead of removing bonelaterally and exploring adjunctive use of mitomy-cin C or by using small shunts around the opticnerve [10,11]. Our preliminary data on cadavertissue suggested that a 6.45-mm wavelength witha 325-mm spot size at 10 Hz produced by theVanderbilt University free electron laser (FEL)might incise the meningeal sheath without dam-aging the underlying optic nerve. No thermaldamage to the optic nerve was evident in this his-tologic study [12]. One group found thermal dam-age at the optic nerve with a contact Nd:YAG la-ser but not with noncontact Nd:YAG or CO2 lasersaimed at adjacent muscle in pigs [13]. These in-vestigators were not attempting to perform opticnerve sheath fenestration. The FEL is an infraredresearch laser that is tunable between 2 and 9 mmwith a 4-msec macropulse structure consisting of atrain of picosecond pulses spaced 350 psec apart.Therefore, wavelengths corresponding to specificmolecular absorption peaks may be selected forinvestigation.

FEL energy tuned at specific wavelengthshas produced minimal collateral damage in someneural and ocular tissues [12,14]. The usefulnessof the Amide II (C-N-H absorption peak) band6.45 mm as an incising wavelength with minimalcollateral damage was demonstrated in brain tis-sue [14]. Besides successfully incising cadavermeningeal sheaths without underlying opticnerve thermal damage at the Amide II wave-length (6.45 mm), incisions with minimal collat-eral thermal damage were produced at the Amide

I band (6.0 mm) for cadaver corneal tissue; and at6.0 mm, 6.1 mm (a water absorption peak), or 6.45mm for cadaver retinal tissue [12]. These datasuggested that the FEL might have a significantadvantage in investigating potential improve-ments in ocular surgery. This opportunity waspossible because glass-hollow waveguides couldbe incorporated into a customized sterilized sur-gical handpiece with 18- to 20-gauge tips for pre-cise delivery of the laser energy to small ocularstructures [15].

The primary purpose of this study was to de-termine whether this FEL Amide II wavelength of6.45 mm [14] is capable of precisely incising theoptic nerve sheath and to compare the histologichealing responses with those produced by tradi-tional mechanical incisions. A second related ob-jective was to understand more about the cell bio-logical response to surgical manipulation of theoptic nerve and to the acute and chronic removalof a portion of the meningeal sheath. Investiga-tors have used MRI to examine optic nerve sheathfenestrations postoperatively [16], but there havebeen no systematic immunocytochemical studiesof the structural changes and glial responses afteroptic nerve sheath fenestration.

METHODS

Animals

Adult Dutch Belted rabbits weighing an av-erage of 2.3 kg were used in this study. All animalprocedures were performed in accordance withNational Institutes of Health and Department ofDefense guidelines and with approval of theVanderbilt Institutional Animal Care and UseCommittee. Rabbits received optic nerve sheathfenestrations with the free electron laser (FEL) (n4 6) or with a knife (n 4 2). Controls involvedeither a sham surgery (n 4 5) or no surgery (n 43). These rabbits survived for 1 month before kill-ing during which time their optic nerve heads andretinas were examined at weekly intervals. Otherrabbits received acute optic nerve sheath fenes-trations with the FEL (n 4 4) or knife (n 4 4) andwere killed immediately after surgery.

Surgical Procedures

Anesthesia was induced with intramuscular(IM) injections of a mixture of ketamine 35 mg/kg,xylazine 5 mg/kg, and acepromazine 0.75 mg/kgwith supplemental half-doses given as needed tomaintain anesthesia. During surgery, the ani-

192 Joos et al.

mals were kept warm and were continuouslymonitored. Under sterile surgical conditions, theeyelids were retracted after making a lateral can-thotomy. Proparacaine drops were instilled in theeye, and a superior conjunctival peritomy wasperformed. A 6-0 Vicryl suture was passedthrough the superior rectus muscle, and thismuscle was disinserted. After extensive retrac-tion the optic nerve could be observed. All eyesexcept unoperated controls were treated with thesurgical steps described above. The surgery wasterminated at this point in the sham controls. Anophthalmic operating microscope was used to vi-sualize the optic nerve sheath and the optic nerveduring all procedures. In 15 experimental eyes, a2-mm-diameter window was produced by incisingthe optic nerve sheath either with a 15 degreeknife or with FEL energy (6.45 mm, 10 Hz, < 2.5mJ, 325-mm spot size, 5-msec macropulse) by us-ing a 530-mm diameter hollow waveguide probe.Energy levels were determined by using an en-ergy meter (Molectron Detector, Inc., Portland,OR) just before and immediately after lasing. Onthe optic nerve sheath of one experimental eye,three times the cutting energy (7.5 mJ) was usedto evaluate the tissue response. The FEL hollowwaveguide delivery system was constructed spe-cifically for these surgical procedures [15]. Withthis system, the FEL beam was transmittedthrough nitrogen-purged pipes to the hollow wa-veguide connector. The hollow waveguide was en-closed within a narrow sterilized surgical probedesigned to be hand held and easy to maneuver.The FEL probe was held approximately 1–2 mmfrom the optic nerve sheath surface. After a cir-cular incision was made in the meninges with ei-ther the FEL or the knife, a Sinsky hook was usedto carefully lift and remove the dura and arach-noid to create the fenestration window. The supe-rior rectus muscle was then reattached. The con-junctiva and the lateral canthotomy wererepaired. The survival rabbits were given Yohim-bine 0.2 mg/kg and Ketoprofen 1 mg/kg IM. Therabbits that received acute procedures were killedwithout awakening from surgery.Ocular Examinations

The survival rabbits were examined weeklywith an indirect ophthalmoscope, and the opticnerve head was photographed and its conditiondocumented.Histology and Immunocytochemistry

The rabbits were given a lethal overdose ofanesthetic and were perfused transcardially first

with PBS followed by 3% formaldehyde, 0.1% glu-taraldehyde (v/v), and 0.2% saturated picric acid(v/v), in 0.1 M phosphate buffer. Eyes with opticnerves attached were removed and placed in thesame fixative overnight. The eyes/nerves thenwere dissected into two components. One compo-nent contained a portion of the retina, optic nervehead, and the segment of optic nerve in which thefenestration had been performed. This componentwas sectioned from superior to inferior. Becausethe fenestration was made close to the globewhere the rabbit optic nerve adheres to the globe,this plane of cut allowed us to identify easily thelocation of the fenestration in sections. The othercomponent contained the proximal segment of op-tic nerve and was cut in cross-section.

The dissected tissue then was dehydrated,embedded in paraffin, and sectioned at 5–10 mm.Selected sections were deparaffinized and stainedwith hematoxylin and eosin (H&E). Serial sec-tions were examined to determine the exact loca-tion of the optic nerve sheath fenestration. Sec-tions through this region then were stained withH&E or immunoreacted for antibodies to glial fi-brillary acidic protein (GFAP) and S100b in se-ries. Comparable regions of optic nerve in allsham or normal controls also were stained or im-munoreacted in the same way.

For immunocytochemistry, the deparaf-finized sections were placed into 100% methanolwith 0.3% hydrogen peroxide (H2O2) to block en-dogenous peroxidase and then were rehydratedand placed into 4% normal goat serum and 0.1%Triton X in Tris buffered saline (TBS). Primaryand secondary antibodies were prepared in TBSwith 0.2% Triton-X 100 and 2% normal horse se-rum. A dilution series was run to determine thebest concentration of primary monoclonal anti-bodies to use for this tissue. S100b (S-2532,Sigma, St. Louis, MO) was used at 1:1,000, GFAP(814-369, Boehringer Mannheim, Indianapolis,IN) was used at 1:500. Negative controls wereperformed by omitting primary antibodies onsome sections in all nerves. No staining was ob-served in control sections. The primary antibodieswere left on the sections overnight, followed bythree rinses in TBS, and then placed in biotinyl-ated horse anti-mouse (BA-2000, Vector, Burlin-game, CA) at 1:200 for 2 hours. Sections wererinsed 3× in TBS and placed in ABC StandardElite (PK-6100,Vector) used at half the recom-mended strength for 2 hours, then rinsed 3× inTBS. Sections were incubated in 0.5% diamino-benzidine (D-5637, Sigma) in TBS with 0.04%

FEL Optic Nerve Sheath Fenestration 193

nickel ammonium sulfate for 15 minutes and thenreacted in the same diaminobenzidine solutionwith 0.006% H2O2 added.

RESULTSSurgical Procedure

Sufficient exposure of the optic nerve was ob-tained in all cases. Once the nerve was exposed

(Fig. 1A), maneuverability with the FEL probewas found to be technically easier in the smallspace compared with maneuverability with theknife. Release of cerebral spinal fluid (CSF) alongthe cut edge of the meninges was taken as theinitial sign that either the laser or knife cut (Fig.1B) was deep enough. Figure 1C shows thewaveguide probe delivering the FEL laser energy

Fig. 1. A: The superior rectus muscle is disinserted and retracted to expose the distal optic nerve sheath in the rabbit. B: A15-degree knife is used to incise the optic nerve sheath. C: A sterile hollow waveguide probe delivers the free electron laser(FEL) energy to the optic nerve sheath. D: A Sinsky hook is used to lift the incised dura and arachnoid layers. E: A windowover the optic nerve is visible after removal of the incised sheath (arrow).

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to the optic nerve sheath. By using the FEL probeviewed under the surgical microscope, it was rela-tively easy and efficient to cut along the circum-ference of a 2-mm diameter circle and then usethe Sinsky hook (Fig. 1D) to lift the dura andarachnoid away from the underlying optic nervefibers. For most rabbit nerves, one pass of thelaser at an average of 2.0 mJ was sufficient to cutthe meninges. Rarely, two passes were required.Several small cuts with the knife were requiredwithin the small retrobulbar space to achieve thesame result achieved in less time with the laserprobe. Initially, a higher energy level of 7.5 mJ(more than 3 times that required to incise themeninges) was tried. This energy was observed toincise too deeply and damage the underlyingnerve, a result that was later confirmed histologi-cally (see below).

Postsurgical Ocular Examinations

No postoperative complications occurred inany of the rabbits. No fundus abnormalities werenoted on weekly indirect ophthalmoscopic exami-nations. Figure 2 shows an example of a fundusphotograph of one rabbit taken 3 weeks after opticnerve sheath fenestration using the FEL. Evenin the case where 7.5 mJ caused histologicallyverified damage to the optic nerve, the retina andoptic nerve head appeared healthy by ophthal-moscopy and not different from normal.

Histology and Immunocytochemistry: Controls

Two types of controls were used in these ex-periments. In three eyes, no surgery was per-formed, and, in 5 eyes, a sham operation was per-formed. Both controls survived for 1 month. Inunoperated controls, glial nuclei showed an evendistribution within the nerve with H&E stain,and the meninges exhibited a normal laminar ap-pearance at the edge of the nerve (Fig. 3A). As-trocytes labeled with either GFAP (Fig. 3B,D) orS100b (Fig. 3C) were relatively evenly distributedthroughout the nerve. In contrast to the normalnerves, all of the sham-operated control nervesexhibited a mild astroglial hypertrophy with fineglial processes at the edge of the nerve which wasexposed during the sham surgery (Fig. 4B,D).This reaction was only evident in astrocytes in thesections reacted for GFAP (Fig. 4B,D) but not inthose stained for H&E to reveal all glia (Fig. 4A)or S100b to show the distribution of astrocytescontaining this calcium binding protein (Fig. 4C).We also believe this was not an edge artifact, be-cause it was consistently located in the regionwhere the nerve was exposed in all five shamcases, a result not seen in unoperated controls.This result suggests that the glial reaction seen inthe sham nerves was a reflection not of glial pro-liferation, per se, but a change in the distributionor number of intermediate filaments within theastrocytic processes at the edge of the nerve sim-ply in response to the minimal mechanical ma-nipulation.

Histology and Immunocytochemistry:Acute Surgery

Sections of the optic nerves through the sitesof the fenestrations produced acutely with theknife and with the FEL at 6.45 mm and < 2.5 mJare shown in Figures 5 and 6, respectively. Boththe knife and the laser were effective in cuttingthe sheath without incising the underlying nerve.The cut edge of the sheath is shown toward thetop of each photomicrograph in subsequent fig-ures. Arrows in Figures 5A (knife) and 6A (laser)showing H&E-stained sections indicate edges ofthe cut sheath. The distribution of glia and sup-porting cells within the nerve revealed in H&Esections was even after acute fenestration witheither the knife (Fig. 5B) or the laser (Fig. 6B),indicating that glia did not divide within the 1- to2-hour period after surgery. However, even dur-ing this short period, a mild hypertrophy of astro-cytes is evident after either the acute knife or la-

Fig. 2. Ophthalmoscopic examination of the fundus demon-strated no change after free electron laser optic nerve sheathfenestration. This photograph was taken 3 weeks after sur-gery.

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ser fenestration as can be seen in the GFAP-immunoreacted sections shown in Figure 5C,D forthe knife and in Figure 6C,D for the FEL. Thisglial reaction was limited to a zone adjacent to thefenestration and was seen after either treatment.The variation in glial hypertrophy seen in theacute fenestration cases with GFAP was withinthe range of that seen after 1-month survival (seebelow). There was no evidence of a change in thedistribution of astrocytes labeled with S100b afteracute fenestration with either the knife (Fig.5E,F) or the FEL (Fig. 6E,F).

Histology and Immunocytochemistry:Chronic Surgery

Damage to the optic nerve only occurred withthe FEL at an excessive energy level (7.5 mJ) as

can be seen in Figure 7 for a rabbit that survivedfor 1 month after the FEL fenestration. In thiscase, thermal damage was clearly evident in theH&E-stained sections through the level of the fen-estration (Fig. 7A,B). As can be seen in Figure 7B,fibrous scar tissue covered the damaged portion ofthe nerve and some fibroblasts appeared to in-vade the edge of the nerve itself. GFAP-positiveastrocytes showed marked hypertrophy immedi-ately adjacent to the fenestration (Fig. 7C,D) andappeared hypertrophied through the full thick-ness of the nerve (Fig. 7D). Astrocytes immunola-beled with S100b were not evident immediatelyadjacent to the site of the fenestration in this casebut were present in neighboring areas (Fig. 7E,F)where S100b appeared to be up-regulated in somecells in comparison to control cells (Figs. 3C, 4C).

Fig. 3. An unoperated control is cross-sectioned to demonstrate the normal distal optic nerve and overlying sheath histology.A: Glial nuclei showed an even distribution within the nerve in an hematoxylin and eosin stain, and the meninges exhibiteda normal laminar appearance at the edge of the nerve. B: Glial fibrillary acidic protein–labeled astrocytes were evenlydistributed. The arrow indicates the same location as in D. C: Astrocytes labeled with S100b were evenly distributed through-out the nerve. D: Higher magnification photomicrograph of B. Scale bars 4 100 mm in B (applies to A–C); 50 mm in D.

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In all other chronic cases, the healing re-sponse after the knife incisions (Fig. 8) and the <2.5 mJ FEL incisions (Fig. 9) appeared quite simi-lar. In H&E-stained sections, a thin fibrous scarwith granulation was evident at the level of thefenestration completely covering the originalopening and extending into the subarachnoidspace (Figs. 8A,B, 9A,B).

In H&E-stained sections, glial and support-ing cells were evenly distributed. However, bothwith the knife (Fig. 8C,D) and with the FEL (Fig.9C,D) fenestrations, astrocytes appeared hyper-trophied in all GFAP-immunostained sections ad-jacent to the original site of the fenestration. Un-like in the acute fenestration cases, in the chronic

cases, we did see evidence of up-regulation ofS100b in glia close to the site of the original fen-estration (Figs. 8E,F, 9E,F). It is not clear wheth-er up-regulation of intermediate filaments(GFAP) occurs in the same glial cells as up-regulation of the calcium binding protein (S100b).The distributions appeared to differ somewhat,suggesting that these responses may involve dif-ferent populations of astrocytes.

DISCUSSION

Our chief finding is that the FEL with pa-rameters of 6.45-mm, < 2.5 mJ, 10 Hz, 5-msec

Fig. 4. A sham surgical control is cross-sectioned 1 month postoperatively to demonstrate the normal distal optic nerve andoverlying sheath histology. A: Glial nuclei showed an even distribution within the nerve in a hematoxylin and eosin stain, andthe meninges exhibited a normal laminar appearance at the edge of the nerve. B: All of the sham-operated control nervesexhibited mild glial fibrillary acidic protein (GFAP) -positive astroglial hypertrophy at the edge of the nerve that was uncov-ered during the sham surgery (arrow). This reaction was only evident in astrocytes in the sections reacted for GFAP. C:Astrocytes labeled with S100b were evenly distributed throughout the nerve. D: Higher magnification photomicrograph of B.Scale bars 4 100 mm in B (applies to A–C); 50 mm in D.

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Fig. 5. Illustrated are sections of the optic nerve through the level of the fenestrations produced acutely with the knife. Theknife was effective in cutting the sheath without damaging the underlying nerve. B, D, and F show higher magnificationphotomicrographs of sections illustrated in A, C, and E, respectively, in this and all subsequent figures. A: Edges of the cutsheath were easily observed (arrows, hematoxylin and eosin [H&E]). B: Distributions of glia and supporting cells within thenerve were uniform after acute fenestration with the knife (H&E). C: A mild hypertrophy of astrocytes was evident after theacute knife fenestration in glial fibrillary acidic protein (GFAP) -immunoreacted sections but was limited to a zone adjacentto the fenestration (arrow). Note that the gap in the section is a histologic artifact. D: Mild GFAP-astrocytic hypertrophy(arrow). E: There was no evidence of a change in the distribution of astrocytes labeled with S100b after acute fenestration withthe knife. F: Astrocytes labeled with S100b. Scale bars 4 100 mm in E (applies to A,C,E); in F (applies to B,D,F).

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Fig. 6. Illustrated are sections of the optic nerve through the level of the fenestrations produced acutely with the free electronlaser (FEL) at 6.45 mm and < 2.5 mJ. The FEL was effective in cutting the sheath. A: Edges of the cut sheath were easilyobserved (arrows; hematoxylin and eosin [H&E]). B: Distributions of glia and supporting cells within the nerve were uniformafter acute fenestration with the laser (arrow; H&E). C: A mild hypertrophy of astrocytes was evident after the acute laserfenestration in glial fibrillary acidic protein (GFAP) -immunoreacted sections, but was limited to a zone adjacent to thefenestration (arrows). D: Mild GFAP-astrocytic hypertrophy (arrows). E: There was no evidence of a change in the distributionof astrocytes labeled with S100b after acute fenestration with the laser. F: Astrocytes labeled with S100b. Scale bars 4 100mm E (applies to A,C,E); in F (applies to B,D,F).

FEL Optic Nerve Sheath Fenestration 199

Fig. 7. Damage to the optic nerve only occurred with the free electron laser at an excessive energy level (7.5 mJ) in a rabbitthat survived for 1 month. A: Damage was evident in the hematoxylin and eosin (H&E) sections through the level of thefenestration. B: Fibrous scar tissue covered the damaged portion of the nerve and some fibroblasts appeared to invade the edgeof the nerve (arrows; H&E). C: Glial fibrillary acidic protein (GFAP) -positive astrocytes were present immediately adjacentto the fenestration and appeared hypertrophied through the full thickness of the nerve. D: Hypertrophied astrocytes (arrows,GFAP). E: S100b–up-regulated cells were present away from the fenestration site (arrows). F: S100b–up-regulated cells werepresent away from the fenestration site (arrows). Scale bars 4 100 mm E (applies to A,C,E); in F (applies to B,D,F).

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Fig. 8. Healing responses after a knife fenestration after 1-month survival are shown. A: A thin fibrous scar was evident atthe level of the fenestration completely covering the original opening and extending into the subarachnoid space (arrow). Noincision of the optic nerve was observed (hematoxylin and eosin [H&E]). B: Glial and supporting cells were evenly distributed(H&E). C: Astrocytes appeared hypertrophied in glial fibrillary acidic protein (GFAP) -immunostained sections adjacent to theoriginal site of the fenestration (arrows). D: Astrocytes appeared hypertrophied in GFAP-immunostained sections adjacent tothe original site of the fenestration (arrows). E: Up-regulation of S100b in some glia near the fenestration site was present(arrows). F: Up-regulation of S100b in some glia near the fenestration site was present (arrows). Scale bars 4 100 mm E(applies to A,C,E); in F (applies to B,D,F).

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Fig. 9. Healing responses after laser fenestration < 2.5 mJ after 1-month survival are shown. A: A thin fibrous scar wasevident at the level of the fenestration completely covering the original opening and extending into the subarachnoid space(arrow). No incision of the optic nerve was observed (hematoxylin and eosin [H&E]). B: Glial and supporting cells were evenlydistributed (H&E). C: Astrocytes appeared hypertrophied in glial fibrillary acidic protein (GFAP) -immunostained sectionsadjacent to the original site of the fenestration (arrows). D: Astrocytes appeared hypertrophied in GFAP-immunostainedsections adjacent to the original site of the fenestration (arrows). E: Up-regulation of S100b in glial cells near the fenestrationsite was present (arrows). F: Up-regulation of S100b in glial cells near the fenestration site was present (arrows). Scale bars4 100 mm E (applies to A,C,E); in F (applies to B,D,F).

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macro pulse, and 325-mm spot size is capable ofefficiently performing an optic nerve sheath fen-estration in a small space. Surgical ease suggeststhat the FEL is superior to the knife in terms ofconvenience and control. Both 6.45-mm FEL inci-sions at < 2.5 mJ and knife incisions result in asimilar rapid hypertrophy of astrocytes at the siteof fenestration, which remains evident 1 monthpostoperatively, although the optic nerve appearsotherwise undamaged.

Evaluation of the Surgical Approach

We found that the Amide II wavelength pro-duced by the tunable FEL at a relatively low en-ergy <2.5 mJ is adequate for performing the opticnerve sheath fenestration in rabbits. Three timesthis energy at 7.5 mJ proved too strong with ob-servable tissue damage of the underlying opticnerve. This finding suggests that, as with otherforms of laser surgery, precise control of laser pa-rameters is critical for successful outcomes. Theinfrared FEL energy is propagated through a thinhollow waveguide that has been specially coatedto allow energy transmission at this wavelength.The waveguide is enclosed within a surgical probewith a 20-gauge diameter tip and a focusing lensat the end. This arrangement allows maneuver-ability in small areas such as over the surgicallyexposed optic nerve sheath. Results of prelimi-nary optic nerve fenestration experiments inmonkeys suggest that the FEL also improves sur-gical ease of this procedure in primates for whichthe difficulty in visualization of the optic nerve issimilar to that in humans [17]. Ideally, one wouldlike to test the usefulness of the FEL in producinga fenestration in animals in which CSF pressurehas been elevated with increased fluid volumesurrounding the optic nerve. Unfortunately, suchmodels would be difficult to produce underchronic conditions with adequate pain manage-ment. However, the surgical procedure should ac-tually be safer and easier to perform under patho-logic conditions of increased CSF pressurebecause the optic nerve would be more protectedfrom an infrared wavelength’s effects by the extrasurrounding cushion of fluid. The maneuverabil-ity of the thin surgical FEL probe also may lead tofuture advances to reduce the invasive surgicalexposure required currently for this procedure.

The Glial Response

Previously, few detailed studies have sys-tematically examined cellular changes and glialresponses after optic nerve sheath fenestration.

Earlier studies have relied on changes identifiedby only routine H&E histology [18–21]. In con-trast, the normal optic nerve has been studiedextensively in a variety of species as a model tounderstand the cell biology of glia [22–24]. Herewe show that, in rabbits, GFAP is up-regulatedrapidly within astrocytes in response to limitedremoval of the nerve sheath. The glial response islocalized to the site of the fenestration and doesnot differ between nerves in which the fenestra-tion is produced with the knife versus the FEL at6.45 mm at < 2.5 mJ. The acute phase of GFAPup-regulation does not appear to be accompaniedby a similar rapid up-regulation of the calciumbinding protein S100b, known to be a reliablemarker for astrocytes [24,25]. However, after1-month survival, up-regulation of S100b is ob-served in a subpopulation of astrocytes adjacentto the fenestration site produced by either theknife or the FEL. This finding suggests that theglial response to the optic nerve sheath fenestra-tion involves at least two phases, i.e., an earlyphase when astrocytes hypertrophy and a laterphase in which some astrocytes up-regulateS100b. It is noteworthy that mild local up-regulation of GFAP but not S100b was also seenin all five sham-operated nerves 1 month postop-eratively. The histologic glial response was ob-served only on the exposed side of the sham sur-geries, suggesting that optic nerve astrocytes arevery sensitive to minimal manipulation of the op-tic nerve sheath and can exhibit changes at least1 month postoperatively. This result indicatesthat glia can respond to mechanical stimuli, al-though our data do not address this possibilitydirectly. In the future, it will be useful to examinemore carefully the degree to which minimal ma-nipulations of the optic nerve itself are capable ofproducing a glial reaction and whether such a re-action resolves with time.

Three groups have examined the postmor-tem appearance of the optic nerve after opticnerve sheath fenestrations in humans. Histologyof patients surviving 3 weeks to 3 months after a6- to 8-mm linear incision of the lateral opticnerve sheath to reduce papilledema demonstratedgranulation tissue at the site [19]. Histologic ex-amination of an optic nerve with H&E 39 daysafter bilateral inferonasal 3 to 5 mm optic nervesheath slits, performed for chronic papilledemacaused by glioblastome multiforme, demonstrateda patent fistula with formed subarachnoid spacecontaining loose connective and arachnoid tissue[20]. In contrast, histopathologic examination of

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bilateral optic nerve sheath fenestrations per-formed for pseudotumor cerebri 14 days beforedeath (unrelated to the fenestration) showed fi-broblasts and localized proliferation of connectivetissue at the site of the original fenestration [21].In this patient, visual recovery and resolution ofheadaches continued after the fenestration untildeath, suggesting that the surgery was still thera-peutically beneficial despite this connective tissueproliferation at the fenestration site. These histo-logic changes are similar to those observed in the1-month survival rabbits in our study. Likewise,in an experimental study of optic nerve fenestra-tion by using a knife in nine monkeys with in-duced papilledema, connective tissue cellsbridged the fenestration and often obliterated thesubarachnoid space under the incision [18]. Thesecells also were observed to invade the underlyingoptic nerve in two cases [18]. The type of healingresponse and the degree to which the scar blocksCSF outflow may partially explain the variabilityin the long-term success with optic nerve sheathfenestration.

The FEL at 6.45 mm and low energy (< 2.5mJ) has shown promise in its ability to success-fully incise the optic nerve sheath in rabbits with-out cutting the underlying optic nerve. Furtherstudies are under way to evaluate this techniquein a primate optic nerve model where the ana-tomic position and sheath are more similar to thatin human optic nerves [17]. If the FEL is againfound equivalent to or better than standard me-chanical optic nerve sheath fenestration methods,then the FEL’s advantage of requiring only a thindelivery probe can be explored further. Perhapsthe extensive and invasive surgical exposure cur-rently required for optic nerve sheath fenestra-tion could be reduced, especially if the laser probecould be combined with an orbital endoscope forimproved visualization.

Recently, a 0.9-mm-diameter endoscope hasbeen successfully combined with a thin glass-hollow waveguide to perform intraocular endo-scopic goniotomy with the FEL in congenital glau-coma rabbits with edematous corneas. This FELsurgery successfully lowered the postoperative in-traocular pressure for 3 weeks and was compa-rable to endoscopic goniotomy performed with thestandard goniotomy needle [26].

Other surgical fields also have successfullyused the FEL. Recently, a neurosurgeon biopsieda small portion of a patient’s benign brain tumor

with FEL energy at 6.45 mm (M. Copeland, per-sonal communication). A dermatology team hasdetermined that FEL at wavelengths 7.2–7.4 mmand 7.6–7.7 mm may be more efficient than theclinical CO2 laser in producing cutaneous contrac-tion [27]. The FEL at 6.1 mm to 6.45 mm has beenfound to efficiently cut cortical bone with less col-lateral thermal damage than a surgical bone saw[28]. Additional studies are required in these andother areas to fully explore the vast potential ofthe FEL. In the future, it also will be significant todetermine whether laser physicists and engineerscan develop a smaller “table-top” FEL, or eco-nomical lasers that can produce single wave-lengths such as 6.45 mm at clinically useful ener-gies and powers. Such developments will benecessary before useful experimental surgery atthese wavelengths can be incorporated into clini-cal practice.

ACKNOWLEDGMENTS

The authors thank Steve Head, Julia Mav-ity-Hudson, Amy Nunnally, Richard Robinson,Steve Head, Tony Adkins, Mike Slowey, and thestaff at the Vanderbilt Keck Free Electron LaserFacility for their valuable technical assistance.

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