Mesure de la facilité d'évacuation de l'humeur aqueuse après la création d'une fistule intrasclérale par injection de l'acide hyaluronique stabilisé dans les yeux de porc

Thesis

Reference

Mesure de la facilité d'évacuation de l'humeur aqueuse après la création d'une fistule intrasclérale par injection de l'acide hyaluronique

stabilisé dans les yeux de porc

MAVRAKANAS, Nikolaos

Abstract

Le but de traitement du glaucome est de maintenir la morphologie de la papille et de préserver la fonction visuelle du patient et la qualité de vie qui lui est liée. Nous présentons ici une nouvelle technique, d'application simple pour créer une microfistule intrasclérale afin de diminuer la pression intraoculaire. Nous créons pour cela un canal intrascléral en injectant une forme d'acide hyaluronique cohésive, qui est perméable à l'humeur aqueuse (NASHA), pour créer un nouveau passage entre la chambre antérieure de l'oeil et l'espace sous-conjonctival. Notre hypothèse est que l'acide hyaluronique cohésive restera pour plusieurs mois dans le canal intrascléral et ceci permettra aux cellules endothéliales de migrer et couvrir ce nouveau canal.

MAVRAKANAS, Nikolaos. Mesure de la facilité d'évacuation de l'humeur aqueuse après la création d'une fistule intrasclérale par injection de l'acide hyaluronique stabilisé dans les yeux de porc. Thèse de doctorat : Univ. Genève, 2010, no. Méd. 10620

URN : urn:nbn:ch:unige-97918

DOI : 10.13097/archive-ouverte/unige:9791

Available at:

http://archive-ouverte.unige.ch/unige:9791

Disclaimer: layout of this document may differ from the published version.

1 / 1

Section de médecine Clinique Département des Neurosciences et Dermatologie

Service d’Ophtalmologie

Thèse préparée sous la direction du Professeur Avinaom B. Safran

" Mesure de la facilité d’évacuation de l’humeur

aqueuse après la création d’une fistule intrasclérale par injection de l’acide hyaluronique stabilisé dans

les yeux de porc "

Thèse

présentée à la Faculté de Médecine de l'Université de Genève

pour obtenir le grade de Docteur en médecine par

Nikolaos MAVRAKANAS de

Katerini, Grèce

Thèse n° 10620 Genève

2010

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yeυx de porc

Lo Fοcυlt6 de m6decine, sυr le pr6οvis de Monsieυr Avinoοm B. SΑFRΑN, professeυr ordinοire

oυ D6pοrtement des Neυrosciences cllniqυes et Dermαto|ogie, αυtorise l'impression de ^lo pr6sente thδse, sοns pr6tendre pοr ld 6mettre d'opinion sυr les propositions qυi y sont 6nonc6es.

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dans les ''Ιnformations relatives λ |a de Genδve".

pr€cddente

pr€sentation et remplir les

des thδses de

conditiοns €numθrdes

doctorat λ l'Universitd

Je tiens à remercier en tout premier lieu le Docteur Tarek Shaarawy pour l‟inspiration de suivre la recherche en glaucome ainsi que pour son soutien, son encouragement

et ses qualités scientifiques et humaines incomparables.

J‟exprime ma profonde gratitude au Professeur Avinoam B. Safran pour m'avoir donné l'opportunité de travailler dans un service de grande qualité, pour son

ouverture d‟esprit, et son immense connaissance, tant sur le plan théorique qu'expérimental.

Un grand merci au Professeur James S. Schutz pour son soutien inconditionnel, son esprit critique, ainsi que pour les réflexions, les questions et les conseils précieux

qu'il m'a prodigués.

Je remercie vivement le Professeur Douglas H. Johnson(1951-2007), et ses collègues, les Docteurs Cindy Bahler et Cheryl Hann, du Département d‟Ophtalmologie de la Clinique Mayo, pour leur accueil chaleureux et leur enthousiasme de m‟apprendre tous les aspects scientifiques et techniques de la

recherche fondamentale en glaucome.

Enfin, je remercie Anthie pour son soutien et son amour, ainsi que mes parents, mon frère Thomas et mes amis qui, de près comme de loin, m'ont aidé et encouragé tout

au long de ce travail.

RESUMÉ DE LA THÈSE DE DOCTORAT EN FRANÇAIS

Introduction

Le glaucome est une maladie oculaire caractérisée par une atteinte progressive de la tête du nerf optique et une altération consécutive du champ visuel. Le facteur de risque le plus important pour le glaucome est l‟élévation de la pression

intraoculaire. Touchant les deux yeux, le glaucome se traduit au début par une atteinte périphérique du champ visuel, qui peut passer inaperçue par le patient mais qui peut aboutir à la cécité.

Le glaucome est la deuxième cause de cécité dans le monde. Il atteint de 1 à 4 % de la population, a souvent un caractère familial et apparaît généralement après 45 ans. C'est dire l'importance d'en dépister rapidement les premiers symptômes afin d'agir pendant qu'il en est temps.

Le but de traitement du glaucome est de maintenir la morphologie de la papille et de préserver la fonction visuelle du patient et la qualité de vie qui lui est liée. La surveillance de la tension oculaire permet de traiter le glaucome à un stade précoce, avant toute altération de la vision. Le traitement vise à faire baisser la tension

oculaire à l'aide de collyres antiglaucomateux (analogues de prostaglandines, bêtabloquants, α-agonistes, inhibiteurs de l‟anhydrase carbonique). S'il se révèle insuffisant, on peut rétablir l'écoulement de l'humeur aqueuse par la chirurgie (trabéculectomie, chirurgie non-pénétrante, utilisation des tubes/valves

intraoculaires) ou à l'aide du laser (trabéculoplastie). Néanmoins, toutes les formes de traitement actuel ne sont souvent pas efficaces à long terme entraînantes des complications locales et systémiques importantes. Il est donc essentiel de

développer des nouvelles formes de traitement antiglaucomateux, qui soient

efficaces, bien tolérées et accessibles sur le plan financier y compris dans les parties défavorisées du monde.

Nous présentons ici une nouvelle technique, d‟application simple pour créer une microfistule intrasclérale afin de diminuer la pression intraoculaire. Nous créons pour cela un canal intrascléral en injectant une forme d‟acide hyaluronique cohésive, qui est perméable à l‟humeur aqueuse (NASHA), pour créer un nouveau passage entre la chambre antérieure de l‟œil et l‟espace sous-conjonctival. Notre hypothèse est que

l‟acide hyaluronique cohésive restera pour plusieurs mois dans le canal intrascléral et ceci permettra aux cellules endothéliales de migrer et couvrir ce nouveau canal.

Matériels et méthodes

Configuration expérimentale

Les yeux des porcs ont été obtenus dans les 24 heures après leur mort, ont été transférés dans des conditions aseptiques, et utilisés le même jour après un

réchauffement dans un bain de 25^oC. L‟utilisation rapide des yeux a prévenu

l‟apparition d‟un œdème cornéen, permettant une bonne visualisation de la chambre antérieure. Du gel lubrifiant (Methocel 2% Omnivision – Neuhausen) a été appliquée à la surface cornéenne pour éviter une déshydratation durant l‟expérimentation.

Sous contrôle microscopique, la chambre antérieure de chaque œil a été canulée au niveau du limbe avec une aiguille de 30 gauge pour infusion. L‟embout de

l‟aiguille a été positionné à l‟aire pupillaire, juste en dessous de l‟iris. L‟aiguille a été connectée avec une pompe de microsyringe (SP 200i, World Precision Instruments, Inc, Sarasota, FL, USA) par intermédiaire d‟un tube de polyéthylène. La chambre antérieure a été par la suite canulée avec une deuxième aiguille, 25 gauge, qui était connectée par un tube de polyéthylène à un transmetteur de pression électronique (Type BLPR, World Precision Instruments, Inc, Sarasota, FL, USA). Les mesures de pression ont été amplifiées et présentées dans un moniteur de pression (Ape BP-1, World Precision Instruments, Sarasota, FL, USA) et transmises dans un système d‟acquisition des données (LabTrax - World Precision Instruments, Inc, Sarasota, FL, USA). Les données ont été enregistrées et imprimées. Tous les tubes étaient remplis par une solution de DPBS (Dulbecco's phosphate buffered saline) (Grand Island Biological Co., Grand Island, N.Y.). La pression intraoculaire était mesurée en mmHg.

Diamètres des aiguilles et longueur du canal intrascleral

10 yeux ont été testés pour chaque combinaison (6 combinaisons) de diamètre de l‟aiguille (21G or 23G or 27G) et de la longueur du canal intrascleral (4mm or 6mm) pour un total de 60 yeux. 10 yeux supplémentaires ont été utilisés comme groupe de contrôle. Aucun canal intrascleral n‟a été crée dans ces yeux.

Mesure de la facilité d‟évacuation de l‟humeur aqueuse

Après la cannulation de la chambre antérieure, le flux a été ajusté pour obtenir une pression intraoculaire (IOP) de 10 mmHg. Le flux a été par la suite accru pour élever la pression intraoculaire à 20 mmHg. Le flux était encore modifié pour arriver à une pression de 30 mmHg.

La facilité d‟évacuation (C) a été calculée par l‟équation de Goldmann:

C=I/IOP

I= I2-I1 ou I1 et I2 représentent le flux (µl/min)

IOP= P2–P1 ou P1 et P2 représentent la pression intraoculaire pour le flux I1 et I2 respectivement (mmHg)

Création d‟un canal intrascléral et injection du gel NASHA

Quand la pression a été stabilisée à 10 mmHg, une aiguille connectée à une seringue avec du gel NASHA a été insérée à l‟espace sous-conjonctival à 4 ou 6 mm du limbe, pour pénétrer dans la sclère et créer un canal intrascleral débouchant dans le trabeculum. La longueur du canal intrascleral (4mm or 6 mm) a été mesurée avec un compas chirurgical.

Par la suite, l‟aiguille a été lentement retirée et en même temps le NASHA gel a été doucement injecté dans ce nouveau canal intrascléral. Un seul canal intrascléral a été crée pour chaque œil utilisé.

Résultats

Une augmentation significative de la facilité d‟évacuation de l‟humeur aqueuse a été observée entre le groupe contrôle et le groupe des 60 yeux injectés du gel NASHA lors d‟un changement de pression intraoculaire de 10 à 20 mmHg ainsi que de 20 à 30 mmHg (voir tableau ci-dessous). L‟augmentation de la facilité

d‟évacuation était indépendante de la longueur du canal créé ou du diamètre de l‟aiguille utilisée pour un changement de pression intraoculaire de 10 à 20 mmHg (p=0.82) ainsi que de 20 à 30 mmHg (p=0.99).

IOP (mmHg)

Groupe de control (moyenne ± sd)

Groupe injecté

(moyenne ± sd) p 95% C.I. pour la différence des moyennes 10-20 0.99 ± 0.13 1.20 ± 0.21 < 0.001 0.11-0,32

20-30 1.07 ± 0.19 1.28 ± 0.18 0.001 0.08-0,33

Conclusion

Nous présentons ici les résultats d‟une nouvelle et simple technique chirurgicale destinée à diminuer la pression intraoculaire qui a été appliquée à des yeux de porc.

Nous avons trouvé que la création d‟un canal intrascleral et l‟injection dans celui-ci de l‟acide hyaluronique cohésive (gel NASHA) permet l‟augmentation de la facilité d‟évacuation de l‟humeur aqueuse, entraînant une réduction de la pression

intraoculaire.

Les avantages de cette technique sont : a) la simplicité et la courte période d‟apprentissage, b) l‟absence d‟hypotonie, rencontrée dans les techniques

chirurgicales de glaucome actuelles, car le gel NASHA crée une barrière mécanique qui empêche la baisse importante de la pression intraoculaire et les complications liées à cette hypotonie, c) la possibilité, si nécessaire, de répéter cette technique pour le contrôle de la maladie.

Il reste à déterminer si les effets de cette technique peuvent être reproduits sur une durée significative chez l‟œil humain.

TABLE OF CONTENTS

PART I (GENERAL PART) DEFINITION OF GLAUCOMA SIGNIFICANCE OF GLAUCOMA

THE ANATOMICAL BASIS OF GLAUCOMA

AQUEOUS HUMOUR PRODUCTION: MOLECULAR MECHANISMS &

REGULATION

AQUEOUS HUMOUR COMPOSITION RELATIVE TO BLOOD AQUEOUS HUMOUR OUTFLOW

MECHANISMS OF AQUEOUS HUMOUR OUTFLOW RESISTANCE TREATMENT OPTIONS IN GLAUCOMA

A. Medical Treatment

B. Laser treatment of the trabecular meshwork (laser trabeculoplasty) C. Surgery

1. Trabeculectomy

2. Non-penetrating glaucoma surgery ( NPGS): deep sclerectomy and viscocanalostomy

3. Drainage implants (tubes and valves)

GLAUCOMA AND WOUND HEALING

THE NEED FOR A SIMPLE, SAFE AND EFFECTIVE PROCEDURE

A new, safer, easier, minimally invasive technique: intrascleral canal creation using modified hyaluronic acid

HYALURONIC ACID Chemical structure Molecular weight Concentration Metabolism

Physiological function

Biocompatibility

NASHA^R: NON-ANIMAL STABILIZED HYALURONIC ACID Starting material

Stabilization and manufacturing Biocompatibility

Residence time of NASHA Gels of any shape and form Clinical use of NASHA

PART II (SPECIFIC STUDY)

AIM OF THE STUDY

MATERIAL AND METHODS Experimental set-up

Needle diameters and canal lengths Outflow facility measurements

NASHA gel ab externo intrascleral injection Statistical analysis

RESULTS DISCUSSION LITERATURE

PART I (General Part)

DEFINITION OF GLAUCOMA

Glaucoma is a progressive optic neuropathy with visual field loss and

characteristic structural changes including thinning of the retinal nerve fibre layer and excavation of the optic nerve head¹ (Figure 1). Intraocular pressure (IOP) elevation is the major risk factor for glaucoma but does not define glaucoma since many patients with glaucoma have IOP measurements that are considered within normal limits. Two major forms of glaucoma exist: open-angle glaucoma, in which aqueous humour has free access to the trabecular meshwork (the aqueous outflow site in the anterior chamber), and angle-closure glaucoma, in which access of the aqueous humour to the trabecular meshwork is anatomically obstructed.

Figure 1. Healthy optic nerve head of the left eye with a small central cupping (left). Note the enlarged cupping (central pale zone) with focal neuroretinal rim thinning at the temporal inferior sector (right).

SIGNIFICANCE OF GLAUCOMA

Glaucoma is the second leading cause of blindness worldwide. Population-based studies have shown that the prevalence of glaucoma is between 2.1% - 4,2% (Table 1) and is more frequent in Asian and African populations (Figure 2)^2-6. A troublesome, yet consistent finding across these studies is that a large portion of glaucoma

worldwide remains undiagnosed. In developed nations, 50% of those with glaucoma

do not know they have it ^{3, 7, 8},a percentage that rises to 62–75% in Hispanic populations within the United States^{5, 9},and to over 90% in developing nations with poor access to health-care^{2, 6}.

Study (Country) Roscommon West Ireland

Kongwa Tanzania

Proyecto Ver United States

Aravind India

Tanjong Pagar Singapore Ethnicity European African Latino South

Asian Chinese

Age range (years) 50+ 40+ 40+ 40+ 40-79

Prevalence: all glaucoma

(%) 2.4 4.2 2.1 2.6 3.7

Prevalence: POAG (%) 1.9 3.1 2.0 1.7 1.8

Prevalence: PACG (%) 0.1 0.6 0.1 0.5 1.1

Prevalence: Ocular hypertension (%) (IOP

cutoff)

4.2 (22) 2.7 (24) 2.3 (22) 1.1 (22) N/A

Table 1. The prevalence of open-angle and angle-closure glaucoma in population-based studies^2-6.

Figure 2. Age-specific prevalence of open-angle glaucoma by ethnicity. (from Quigley HA, Broman AT. The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol. 2006;90:262–7.)

The frequency of bilateral blindness amongst patients with glaucoma varies across populations2-4, 6, 9 -14

,however there is increased bilateral blindness from glaucoma in developing countries with poor access to ophthalmic care^{2, 6}, and in populations where angle-closure glaucoma predominates4, 10, 11, 13 (Table 2).

Study Country % of glaucoma patients bilaterally blind Open-angle glaucoma

Los Angeles Latino Eye Study USA 1.0

Beaver Dam Eye Study USA 2.5

Baltimore Eye Study USA 4.4

Roscommon Eye Study Ireland 7.3

Aravind Comprehensive Eye Study India 9.4

Kongwa Eye Study Tanzania 9.6

Angle-closure glaucoma

Northwest Alaska Eskimo USA 20.0

Hövsgöl Mongolia 21.4

Andhra Pradesh Eye Study India 25.0

Tanjong Pagar Singapore 35.7

Table 2. Frequency of bilateral blindness in populations with open-angle and closed-angle glaucoma

Demographic projections with prevalence models for open-angle and angle- closure glaucoma estimate that 61 million people worldwide will have glaucoma by 2010, and 8.4 million will be bilaterally blind from the disease¹⁵ (Table 3). Growth and aging of the world‟s population are expected to result in significant increases in these numbers. While primary angle closure glaucoma (PACG) remains less common than primary open angle glaucoma (POAG), the numbers of individuals expected to be blind from both types of glaucoma is nearly equal given the higher morbidity of PACG.

Year POAG Blind POAG PACG Blind, PACG Total Blind, Total

2010 45 4.5 16 3.9 61 8.4

2020 59 5.9 21 5.3 80 11.2

Table 3. Projections of total numbers of patients (in millions) with POAG and PACG and of resultant associated blindness

THE ANATOMICAL BASIS OF GLAUCOMA

The two main structures related to aqueous humour dynamics are the ciliary body, the site of aqueous production, and the limbal region, which includes the main site of aqueous egress, the trabecular meshwork. Figure 3 shows the relationship between these 2 structures and the surrounding anatomy:

1. The limbus is the transition zone between the cornea and the sclera. On the inner surface of the limbus is an indentation, the scleral sulcus, which has a sharp posterior margin, the scleral spur, and a sloping anterior wall that extends to the peripheral cornea.

Figure 3. Anatomy of the angle (from Wolff's Anatomy of the Eye and Orbit, A. J. Bron, R. C.

Tripathi, B. J. Tripathi eds. A Hodder Arnold Publication; 8th edition, 1997)

2. The trabecular meshwork is a sievelike structure, which bridges the scleral sulcus and leads into a tube, the Schlemm‟s canal. Schlemm‟s canal is an endothelial- lined channel which runs 360-degrees around and within the limbus. It is often single but occasionally branches into a plexus-like system. Schlemm‟s canal is connected by fine intrascleral channels to the episcleral veins. The trabecular

meshwork, Schlemm‟s canal, and the intrascleral channels comprise the main route of aqueous humour outflow. The meshwork begins anteriorly at the periphery of the cornea at a ridge known as Schwalbe‟s line.

Figure 4. The three layers of trabecular meshwork: uveal, corneaoscleral, and

juxtacanalicular (From Shields‟ Textbook of Glaucoma, Lippincott Williams & Wilkins 2005).

The trabecular meshwork (Figure 4) can be divided into three portions: (a) uveal meshwork, (b) corneoscleral meshwork, (c) juxtacanalicular meshwork. The uveal meshwork is the inner most portion of the trabeculum, adjacent to the aqueous humour in the anterior chamber. It is arranged in bands that extend from the iris root and ciliary body to the peripheral cornea. The arrangement of the bands creates openings that vary in size from 25 to 75 µm¹⁶. The corneoscleral meshwork extends from the scleral spur to the anterior wall of the scleral sulcus and consists of sheets

of trabeculae that are perforated by elliptical openings. These holes become progressively smaller as the trabecular sheets approach Schlemm‟s canal, with a diameter range of 5-50µm¹⁶ (Figure 5). The juxtacanalicular meshwork consists of three layers, an inner endothelial layer, a central connective tissue layer and an outer endothelial layer (also inner wall of Schlemm‟s canal), which is the last tissue

aqueous humour must traverse before entering the Schlemm‟s canal¹⁷.

Figure 5. Schematic of aqueous humour outflow into the Schlemm‟s canal. (From Hogan MA, Alvarado J, weddell J. Histology of the human eye. Philadelphia: WB Saunders, 1971) .

3. The ciliary body attaches to the sclera spur on cross section, and has a triangular shape. The ciliary processes (the actual site of aqueous humour production)

occupy the inner and anterior most portion of the ciliary body in a structure called the pars plicata. The pars plicata region also contains smooth muscle which serves the important function of accommodation. The ciliary processes consist of 70-80 radial ridges (major processes), between which are interdigitated an equal number of smaller ridges (minor processes). The posterior portion of the ciliary body, called the pars plana, has a flatter inner surface and joins the choroid at the ora serrata (Figures 6, 7).

Figure 6. Anterior segment of the eye viewed from behind (from Atlas of human anatomy, F. H. Netter, Rittenhouse Book Distributors Inc.; 2nd edition, 1997).

Figure 7. Ciliary body viewed from behind. The arrow points on a small ciliary process (from Atlas of human anatomy, F. H. Netter, Rittenhouse Book Distributors Inc.; 2nd edition, 1997).

The ciliary processes are composed of (a) capillaries, (b) stroma and (c) epithelia. Two layers of ciliary epithelium surround the stroma, with the apical surfaces of the two cell layers in apposition to each other: the pigmented epithelium which has numerous melanin granules and an atypical basement membrane on the stromal side and the non pigmented epithelium, which has a basement membrane composed of glycoproteins that are immunoreactive for laminin and collagens I, III and IV¹⁸. This membrane which faces the aqueous humour is also called the internal limiting membrane and fuses with the zonules.

A variety of intracellular junctions connect adjacent cells within each epithelial layer (Figure 8), as well as apical surfaces of the two layers^19,20,21 including (a) gap junctions²², which are expressed between pigmented cells, non pigmented

cells and pigmented-non pigmented cells and (b) tight junctions or zonulae

occludens, which are expressed between the non-pigmented cells¹⁹. It is primarily the zonulae occludens in the non pigmented ciliary epithelium that creates an effective barrier (blood aqueous barrier) to intermediate and high molecular weight substances, such as proteins.

Figure 8. Schematic of the ciliary epithelium summarizing the histology and junctional complexes (From Shields‟ Textbook of Glaucoma, Lippincott Williams & Wilkins 2005).

4. The iris inserts into the anterior side of the ciliary body, leaving a variable width of the ciliary body visible between the iris root and the scleral spur, referred to as the ciliary band. The iris separates the aqueous humour compartment into a posterior and anterior chamber, and the angle formed by the iris and the cornea is called the anterior chamber angle.

Figure 9. Lens is suspended from the ciliary body by zonules (from Wolff's Anatomy of the Eye and Orbit, A. J. Bron, R. C. Tripathi, B. J. Tripathi eds. A Hodder Arnold Publication;

8th edition, 1997).

5. The lens is suspended from the ciliary body by zonules and separates the vitreous posteriorly from the aqueous humour anteriorly (Figure 9).

AQUEOUS HUMOUR PRODUCTION: MOLECULAR MECHANISMS AND REGULATION

Aqueous humour is a dynamic intracellular fluid that is vital to the health of the eye. Using ocular fluorophotometry, the mean rate of aqueous humour inflow is 2.97 μL/min between 8 a.m. and noon, 2,68 μL/min the afternoon and 1,28 μL/min

between midnight and 6 a.m. ²³. These changes in aqueous humour flow throughout the day reflect a biological pattern, as circadian rhythm. This circadian pattern in flow rate accounts for a similar circadian pattern of IOP.

The precise location of aqueous humour production appears to be in the non pigmented ciliary epithelium, of the anterior portion of the pars plicata along the tips and crests of the ciliary processes. These cells have increased basal and lateral interdigitations, mitochondria, and rough endoplasmic reticulum fenestrated capillary endothelium, a thinner layer of ciliary stroma, and an increase gap junctions between pigmented and non pigmented epithelia²⁴.

Aqueous humour is derived from plasma within the capillary network of the ciliary processes. The circulating aqueous humour enters into the posterior cha mber and flows around the lens and through the pupil into the anterior chamber. A temperature gradient (cooler aqueous toward the cornea) creates a flow pattern, which may be visualised in patients with inflammatory cells in the anterior chamber. Constitutents of aqueous humour enter the posterior chamber via the non pigmented epithelium by the following processes:

a. Diffusion. Lipid soluble substances are transported through the lipid portions of the membrane proportional to a concentration gradient across the membrane.

b. Ultrafiltration. Water and water-soluble substances, limited by size and charge, flow through micropores in the protein of the cell membrane in response to an osmotic gradient and hydrostatic pressure.

c. Secretion. Substances of larger size or greater charge are actively transported across the cell membrane. Secretion is mediated by transporters, membrane proteins, which consume energy generated by adenosine triphosphate (ATP) hydrolysis.

Although the exact contributions of these three processes in aqueous humour production are not known, it is thought that active secretion accounts for 80% -90% of total aqueous humour formation²⁵.

AQUEOUS HUMOUR COMPOSITION RELATIVE TO BLOOD²⁵ 1. Slightly hypertonic

2. Acidic

3. Ascorbate excess 4. Protein deficit

5. Slight excess of chloride and lactic acid

6. Slight deficit of sodium, bicarbonate, carbon dioxide and glucose

7. Other reported constituents and features: amino acids, sodium hyaluronate, norepinephrine, coagulation properties, tissue plasminogen activator, collage nase activity

AQUEOUS HUMOUR OUTFLOW

Most of the aqueous humour leaves the eye at the anterior chamber angle

through the trabecular meshwork, and then through the Schlemm‟s canal, intrascleral channels, and episcleral and conjunctival veins. This primary outflow pathway is referred to as the conventional or trabecular outflow. In the unconventional or uveoscleral outflow, aqueous humour exits by passing through the root of the iris, between the ciliary muscle bundles, and then through the suprachoroidal and scleral tisssues^26,27,28.

Trabecular outflow accounts approximately for 90% of the aqueous human egress from the eye in humans, with the remaining 10% corresponding to the uveoscleral outflow. Whereas both trabecular and uveoscleral outflow facility decrease with age, there is a relative increase in the contribution of trabecular outflow in older

eyes^26,27,28.

From the anterior chamber to the Schlemm‟s canal, aqueous humour passes easily through the openings of the uveal and corneoscleral meshwork but finds the most important resistance while traversing the juxtacanalicular portion of the

trabeculum. Openings in the inner wall endothelium of Schlemm‟s canal (outer endothelial layer of the juxtacanalicular meshwork) consist of minute pores and giant vacuoles²⁹ (Figures 10, 11) which vary from 0.5 to 2.0 µm^16,30,31. Although early studies suggested that these structures were artifacts, it is now believed that they are physiologic structures involved in the transcellular transport of aqueous humour²⁹. Evidence in support of their role in the transcellular outflow is based on the injection of tracer elements into the anterior chamber with the demonstration of the tracer in the vacuoles and pores^32,33,34. The observation that the concentration of the tracer material in the giant vacuoles is not always the same as in the juxtacanalicular

connective tissue implies a dynamic system in which the vacuoles intermittently open and close to transport aqueous to the Schlemm‟s canal³³. Whether this transcellular transport is active or passive is controversial. Indirect evidence for active transport includes the presence of enzymes³⁵ and microscopic structures³⁶ compatible with an active transport system in the endothelial juxtacanalicular layer. However, the bulk of evidence supports the theory of passive (pressure-dependent) transport because the size and number of vacuoles increase with progressive IOP elevation^37,38. In addition, this phenomenon is reversible in enucleated eyes, and hypothermia has no effect on the development of vacuoles in the enucleated eye^39,40. An alternative theory to that of transcellular transport is paracellular transport between the inner wall endothelial cells.

Figure 10A shows a light microscopic view of Schlemm‟s canal (SC) and adjacent trabecular meshwork in a monkey eye. Trabecular wall of Schlemm‟s canal (TW) with prominent vacuolated cells (arrows); corneoscleral wall of Schlemm‟s canal (CW);

collector channel (CC). Figure 10B is an electron microscopic view of trabecular wall of Schlemm‟s canal, showing vacuolated endothelial cells (V) containing flocculent material (FL). OZ, occluding zonules, BM, basement membrane, OS, open spaces in endothelial meshwork.

Figure 10 Ultrastructure of Schlemm's canal in a monkey‟s eye in relation to aqueous outflow. (From Tripathi RC. Exp Eye Res. 1968 Jul;7(3):335–341).

Figure 11. Theory of transcellular humour transport in which series of pores and giant vacuoles open on connective tissue side of juxtacanalicular meshwork. Fusion of basal an d apical cells creates a temporary transcellular channel that allows flow of aqueous humour into Schlemm‟s canal (from Tripathi RC. Mechanism of the aqueous outflow across the trabecular wall of Schlemm's canal. Exp Eye Res, 1971;11:116-21).

MECHANISMS OF AQUEOUS HUMOUR OUTFLOW RESISTANCE A. Resistance in the trabecular meshwork

Although the precise mechanism of resistance to conventional outflow is not clearly understood, the following observations provide some insight into this important question.

Glycosaminoglycans. Both human and animal studies of trabecular meshwork indicate that the cells synthesize a heterogenous mixture of glycosaminoglycans, which include hyaluronic acids, chondroitin sulphate, keratin sulphate, and heparin sulphate^{41, 42,43}. It has been suggested that enzymes which catabolise

glycosaminoglycans are released by lysosomes in the meshwork and depolymerise glycosaminoglycans, thereby reducing resistance to outflow⁴⁴. However, perfusion studies with enucleated monkey eyes suggest that outflow resistance due to

glycosaminoglycans is probably only slightly relevant to the trabecular meshwork⁴⁵. Glucocorticoid mechanisms. Glucocorticoids inhibit the synthesis of endogenous prostaglandins, which is clinically relevant because prostaglandins increase IOP in high doses⁴⁶ but reduce IOP in moderate and low concentrations⁴⁷. Glucocorticoid receptors have been identified in human trabecular cells^{48, 49} and outflow facility may be influenced by the effect of glucocorticoids on the extracellular matrix metabolism of trabecular cells⁵⁰.

Myocilin. Myocilin is believed to be clinically important in the pathogenesis of some forms of juvenile glaucoma⁵¹. Also some studies have shown increased myocilin expression in trabecular cells in response to dexamethasone, although other studies failed to confirm these findings^{52,53, 54}.

Fibrinolytic activity. Fibrinolytic activity has been demonstrated in the endothelium of Schlemm‟s canal⁵⁵, but no evidence of coagulation factors has been found⁵⁶. Tissue plasminogen activator, which converts plasminogen to plasmin, thereby mediating the lysis of formed fibrin, has been found in many ocular tissues, including the trabecular meshwork⁵⁷. This suggests that a hemostatic balance, displaced towards fibrinolysis, protects this portion of the outflow system from occlusion by fibrin and platelets. Tissue plasminogen activator may also influence outflow 41

resistance under normal circumstances by altering the glucoprotein content of the extracellular matrix⁵⁸.

Pressure dependent changes. Elevation of IOP is associated with increased resistance to aqueous humour outflow^37,59 which appears to be related to a collapse of Schlemm‟s canal. Histologic studies of eyes perfused at different pressures

suggest that compromise of canal lumen with elevated IOP is due to the distention of the trabecular meshwork^37,60,61, an increase in endothelial vacuoles³⁸ and a

ballooning of the inner wall endothelial cells into the canal³⁹. As it is expected by these observations, resistance to outflow is decreased by expanding Schlemm‟s canal. Further evidence for the effect of expanding Schlemm‟s canal may be supported by the IOP-lowering effect of viscocanalostomy⁶². In contrast, after

successful filtration surgery, there is a decrease in the size of Schlemm‟s canal, most likely due to aqueous underperfusion of the meshwork⁶³.

Age-related changes. The normal human trabecular meshwork undergoes several changes with age^{64, 65,66}. The general configuration changes from a long wedge shape to a shorter more rhomboidal form. The scleral spur becomes more prominent, the uveal meshwork more compact and localized closures of Schlemm‟s canal

become more common. The trabecular beams progressively thicken, the endothelial cellularity declines, approximately at the rate of 0,58 % of cells per year and the number of giant vacuoles decreases. A narrowing of intratrabecular spaces and an increase in extracellular material, especially near the juxta-canalicular tissue, are also seen with age.

B. Resistance to Unconventional outflow

The uveoscleral pathway is characterized as „pressure independent‟, is reduced by cholinergic agonists and decreases with aging^28,67. A potential explanation of the uveoscleral outflow decrease with aging is the thickening of elastic fibers in the ciliary muscles⁶⁷. Part of the uveoscleral outflow may be pressure-sensitive as there are reports of increased uveoscleral outflow with elevated IOP, presumably due to

ultrafiltration of the aqueous into uveal vessels^27,68. Uveoscleral outflow is reduced by miotics, and enhanced by prostaglandins.

C. Episcleral venous pressure

The precise relationship between episcleral venous pressure and aqueous humour dynamics is complex and only partially understood. It has been commonly advocated that IOP increases mmHg to mmHg with increased episcleral venous pressure, although it may be that the magnitude of IOP increase is greater than the venous pressure increase⁶⁹. The normal epicsleral venous pressure is reported to be within the range of 8 to 11 mm Hg⁷⁰.

TREATMENT OPTIONS IN GLAUCOMA

The goal of glaucoma therapy is to maintain quality of life by preserving visual function while avoiding therapeutic side effects and complications⁷¹. Elevated intraocular pressure (IOP) is the main and only confirmed risk factor for damage in glaucoma and hence the concept of the need for a specific target pressure in an individual eye⁷². It is clear that lowering IOP by medical or surgical treatment often arrests the progression of glaucomatous visual loss^{73, 74}. Other factors, such as induced apoptosis (e.g., N-methyl-D-aspartate, NMDA, receptor excitotoxicity, nitric oxide synthase dysregulation), and alterations of microcirculation and autoimmune function may have a role in the pathogenesis of glaucoma⁷⁵. Medical and surgical treatments reduce aqueous secretion or increase aqueous outflow.

A. Medical Treatment (Table 4)

Medical treatment with anti-glaucoma medications is helpful for many patients however there are many drawbacks: (1) lack of pressure control despite maximal medical therapy is a problem for many patients, (2) multiple medications are necessary for the lifetime for pressure control in many patients and represents a problem in cost of medication for many of them (3) patient compliance in taking the medications is a problem for many patients, particularly those taking multiple medications including as many as 8 or more drops in each eye at different times daily. Inadequate compliance may be the most serious limiting factor in the

nonsurgical therapy of glaucoma particularly in patients who are on several glaucoma medications simultaneously. Factors that influence compliance include complexity of the medical regimen, side effects of the medications, the cognitive state of the

patient, psychological factors, and patient understanding of the disease and its treatment^{76, 77}. (4) most of the medications have side effects, both ocular and systemic, which range from brow ache and dimming of vision and spasm of accommodation in all patients with miotics and rarely retinal detachment

(parasympathomimetics), ocular redness and vasodilatation and allergic reactions caused by alpha agonists such as epinephrine and dipivefrin, increased iris or dark circles around the eyes, periocular skin pigmentation, as well as eyelash overgrow th with PGAs, decreased pulmonary function, and occasionally depression, asthma, and heart block with β-blocker drops, kidney stones, bad taste, tingling in the hands

and feet, loss of appetite and rarely aplastic anemia with carbonic anhydrase inhibitors such as dorzolamide and adetazolamide. Serious side effects may be related to coincident systemic diseases, drug interactions, and affect the success or failure of medical treatment.

Antiglaucoma Medications TOPICAL

Adrenergic Antagonists β-Blockers

• timolol • levobunolol • betaxolol • carteolol • metipranolol Adrenergic Agonists Nonselective

• epinephrine • dipivefrin α2-Selective

• brimonidine • apraclonidine

Miotics (Direct Parasympathomimetics) • pilocarpine

Prostaglandins/Prostamides

• travoprost, bimatoprost, unoprostone, latanoprost Topical Carbonic Anhydrase Inhibitors

• dorzolamide • brinzolamide Combination Products

• timolol/dorzolamide • timolol/pilocarpine • timolol/latanoprost SYSTEMIC

Carbonic Anhydrase Inhibitors • acetazolamide

• dichlorphenamide • methazolamide Hyperosmotic Agents

• mannitol • glycerol

Table 4. Classification of antiglaucoma medications

B. Laser treatment of the trabecular meshwork (laser trabeculoplasty) Laser trabeculoplasty, laser burns of the trabecular meshwork under

microscopic control, is not usually successful in controlling open angle glaucoma permanently by itself (Figures 12, 13) although it is frequently performed. The Glaucoma Laser Treatment Trial (GLT) compared topical timolol (β-blocker) and argon laser trabeculoplasty (ALT) as initial therapy, and found similar results after 7 years⁷⁸. A full treatment of selective laser trabeculoplasty (SLT), (360°) compared with topical latanoprost as initial treatment showed no difference in IOP control at 1 year of follow-up⁷⁹. However, IOP spikes, hemorrhage, uveitis, scarring of the trabecular meshwork, corneal and lens damage can occur as a result of laser treatment of the trabecular meshwork.

Figure 12. Argon laser trabeculoplasty (from Yanoff & Duker, Ophthalmology, 3^rd edition, Elsevier 2009).

Figure 13. Argon versus selective laser trabeculoplasty. Note the larger SLT spots (from Yanoff & Duker, Ophthalmology, 3^rd edition, Elsevier 2009).

C. Surgery

1. Trabeculectomy

Trabeculectomy (Figure 14) is a surgical procedure to create a fistula from the anterior chamber to the potential subconjunctival space; the fistula is partially protected by a partial thickness scleral flap which offers some resistance to flow in order to try to prevent overfiltration. The goal is for the fistula to form a filtering bleb or conjunctival bulla from which aqueous humour is absorbed into the surrounding ocular tissues thereby reducing IOP. Modalities to alter wound-healing fibrosis in the subconjunctival space, such as antimetabolite (5-fluorouracil and mitomycin C) usage^80-86 and laser suture-lysis⁸⁷ have been used to try to improve postoperative success. However, while trabeculectomy is considered the “gold standard” for glaucoma surgeries there is a very significant complication and failure rate^80-86

Figure 14. Limbal based trabeculectomy (from Yanoff & Duker, Ophthalmology, 3rd ed, Elsevier 2009)

The risks and complications of trabeculectomy include:

 Intraoperative and early postoperative: suprachoroidal haemorrhage, infection, hypotony, shallow chamber, choroidal effusions, bleb leaks, aqueous

misdirection, cataract, discomfort, cystoid macular oedema, astigmatism, hypotony, maculopathy, vision loss, and commonly failure with need for additional surgery.

 Late postoperative: discomfort, aqueous leaks, intraocular infection, failure, cataract development or progression, ptosis, permanent vision decrease or loss, need for additional surgery.

2. Non-penetrating glaucoma surgery - NPGS (deep sclerectomy and viscocanalostomy)

In order to try to reduce complications associated with trabeculectomy surgery, non-penetrating filtering procedures have been developed to reduce intraocular pressure by enhancing the natural aqueous outflow channels reducing outflow resistance located in the inner wall of the Schlemm's canal and the juxtacanalicular trabecular meshwork⁸⁸. In the last few years viscocanalostomy and deep sclerectomy have become the most popular non-penetrating filtering procedures. Both involve removal of a deep scleral flap, the external wall of Schlemm's canal and corneal stroma behind the anterior trabeculum and Descemet's membrane, thus creating an intrascleral space (Figure 15). After aqueous humour passage through the intact trabeculo-Descemet‟s membrane, four mechanisms of aqueous resorption may occur; a subconjunctival filtering bleb, an intrascleral filtering bleb, a suprachoroidal filtration, and an episcleral vein outflow via Schlemm‟s canal⁸⁸.

Figure 15. Creation of a superficial and deep scleral flap (left). Peeling of the inner wall of Sclemm's canal and of juxtacanalicular trabeculum using fine forceps (right)⁸⁸.

Nonpenetrating glaucoma surgery is contraindicated in the presence of narrow angles because of complications of iris incarceration or anterior synechia, thus limiting the potential candidates for these procedures. Contraindications for NPGS not only include narrow-angles, but status post laser trabeculoplasty, post-traumatic angle-recession glaucoma and neovascular glaucoma. The technique is associated with a long learning curve. Published clinical trials comparing nonpenetrating

glaucoma surgery to full-thickness trabeculectomy have a consensus on the superior safety profile of non-penetrating glaucoma surgery but are not in agreement when it comes to efficacy⁸⁸.

3. Drainage implants (tubes and valves)

Drainage implants (Figure 16) are devices that produce a fistula from the acterior chamber via a tube with a pressure regulating valve or a tube with a resevoir. Such implants provide a good surgical option for many eyes in which there is a high probability of trabeculectomy failure from extensive conjunctival scarring and or previous failed procedures or the type of underlying glaucoma (neovascular, uveitic, iridocorneal endothelial (ICE) syndrome, etc). A role for these implants may exist at an earlier stage for some patients, particularly with the

increased concern about postoperative late bleb leaks and risks of late endophthalmitis following the use of antimetabolites; however the surgery is frequently more complex, involves use of a permanent foreign material, and has a significant rate of complications.

A recent trabeculectomy vs tube (TVT) study⁸⁹ reports IOP levels as low as trabeculectomy with tubes but with more postoperative medication needed. Tube surgery had a higher success rate compared to trabeculectomy with mitomycine (MMC) during the first 3 years of follow-up. Both procedures were associated with similar IOP reduction and use of supplemental medical therapy at 3 years. While the incidence of postoperative complications was higher following trabeculectomy with MMC relative to tube shunt surgery, most complications were transient and self-limited.

Figure 16. Silicone tube is inserted through 22- or 23-gauge needle opening at limbus. Note distal end of Supramid suture that protrudes forward beneath anterior cut edge of the

conjunctiva and the tube of the previously placed implant. (from Yanoff & Duker, Ophthalmology, 3rd ed, Elsevier, 2009).

Intraoperative complications include bleeding, misdirection of silicone tube, and loss of anterior chamber. Early postoperative complications include hypotony, with or without associated choroidal effusions. Hemorrhagic effusions are often

associated with pain, even though the eye may remain hypotonic. Cataract and infection may occur. Tube-corneal contact may occur leading to corneal

decompensation. Occlusion of the tube by vitreous may occur in aphakic eyes.

Late complications include the development of a thick capsule around the plate, which results in an elevation of IOP, erosion of the silicone tube through the sclera or scleral patch and conjunctiva, plate migration, limitation of eye movement, infection, retinal detachment, sterile hypopyon, and infection.

GLAUCOMA AND WOUND HEALING

The healing process after glaucoma filtration is the main determinant of surgical failure. The ability to fully control wound healing may ultimately give us the ability to control the intraocular pressure for all patients undergoing glaucoma filtration surgery. Tissue trauma and inflammation stimulate the wound healing, which is classically divided in the inflammatory, proliferative and remodeling phase.

In the inflammatory phase clotting takes place in order to obtain hemostasis, and various factors like serotonin, bradykinin, prostaglandins, prostacyclins, thromboxane, and histamine are released to attract cells (polymorphonuclear neutrophils and macrophages) that phagocytise debris and damaged tissue. In the next phase, proliferation, fibroblasts begin to enter the wound site, even before the inflammatory phase has ended. Angiogenesis, fibroplasia and granulation tissue formation, collagen deposition, epithelialization and contraction are the major events during this phase, which is also called the reconstruction phase. VEGF and TGF play an important role during this stage.

During the last phase, remodeling, type III collagen, which is prevalent during proliferation, is gradually degraded and the stronger type I collagen is laid down in its place. Originally disorganized collagen fibers are rearranged, cross-linked, and aligned along tension lines. This phase may last for a year or longer.

In table 5, the sequence of events in wound healing and potential areas of modification after glaucoma filtering surgery are summarised.

Table 5.

Sequence of events in wound healing and potential areas of modification after glaucoma filtering surgery are summarised. (Modified from Khaw PT, et al.

Modulation of wound healing after glaucoma surgery. Curr Opin Ophthalmol.

2001;12(2):143-8).

.

THE NEED FOR A SIMPLE, SAFE AND EFFECTIVE PROCEDURE

Surgical techniques for lowering intraocular pressure (IOP) have evolved since the first trabeculectomy was performed in late 1960s^{90, 91}. Non penetrating glaucoma surgery (NPGS)⁹² and shunt tubes have been shown to be useful in the surgical treatment of glaucoma⁹³ and trabeculectomy still remains the procedure of choice for most surgeons around the world⁹⁴. However, long term efficacy of trabeculectomy and tube shunts as well as complications^{95, 96}, and suboptimal degree and longevity of IOP-reduction with less invasive techniques⁹⁷ indicate the need for new, safer, effective glaucoma surgery. A safe, simple, effective surgical treatment would be an attractive option for patients burdened by expensive medical glaucoma treatment, patients with compliance problems, and for patients in developing countries for whom medical treatments are unavailable or inaccessible⁹⁸.

A new, safer, easier, minimally invasive technique: intrascleral canal creation using modified hyaluronic acid

Searching for a new, simple, reliable surgical technique to lower intraocular

pressure, we investigated the creation of a microfistula between the anterior chamber and the potential subconjunctival space in isolated porcine eyes by injecting

intrasclerally stabilized, non-animal, hyaluronic acid (NASHA™, Q-Med AB, Uppsala, Sweden). Several combinations of intrascleral canal lengths and needle diameters were used in this experiment. Aqueous humour passes though the NASHA™ gel in the scleral canal, creating a new aqueous outflow pathway. The choice of hyaluronic acid to fill the scleral canal in these experiments was based on the particular, special characteristics of this biomolecule.

HYALURONIC ACID

Meyer and Palmer isolated the polysaccharide hyaluronic acid (sodium hyaluronate, hyaluronan) in 1934 from the vitreous of bovine eyes. They found a substance, which contained two sugar moieties, one of which was uronic acid.

Therefore, to cite the authors, “we propose, for convenience, the name „hyaluronic acid‟, from hyaloid (vitreous) + uronic acid”. Under physiological conditions the polysaccharide is not present in the acid form but exists as a salt: hyaluronate. The most abundant cation in tissues is sodium, and hyaluronic acid is generally present as sodium hyaluronate both in tissues and in products.

Chemical structure

Hyaluronic acid has a simple chemical structure: a disaccharide unit containing glucuronic acid and N-acetylglucosamine. These are joined together forming a uniform, linear polysaccharide molecule as shown in the following figure:

Figure 17. Hyaluronic acid chemical structure.

The number of repeating disaccharide units is denoted by “n”. These sugar units are hydrophilic. Water is attracted to hyaluronic acid, which is therefore highly soluble in water. Hyaluronic acid contains these, and only these, two sugar units in all tissues and in all species. The identical hyaluronic acid molecule can also be manufactured from a non-animal source by modern biotechnological methods. Hyaluronic acid regardless of method of synthesis only contains the simple disaccharide units without amino acids, proteins or other sugar moieties.

Molecular weight

Hyaluronic acid is a uniform, linear and unbranched molecule consisting of multiple identical disaccharide units so that the only variation between various hyaluronic acid preparations is the length and size of the polymer. For example, the molecular size of hyaluronic acid is often lower in synovial fluid from patients with joint disorders. In healthy tissues the molecular weight of hyaluronic acid is typically in the order of 5 to10 million. In some tissues or species, especially in diseased tissues, the molecular weight may be lower: ~1 million. The molecular weight of synthetic hyaluronic acid products varies from 0.5 to 5 million. For comparison, the molecular weight of typical proteins is <100 000. Most polysaccharides in vertebrates have a molecular weight in the order of 10 000 and are linked to various types of proteins

Concentration

Hyaluronic acid is an essential component of the extra cellular matrix of all tissues. Especially high concentrations are found in tissues such as the umbilical cord (4 mg/g), synovial fluid (3-4 mg/ml) and vitreous (0.1-0.4 mg/g). The average concentration of hyaluronic acid in the human body is 200 mg/kg (0.02%). Thus, a human body weighing 60 kg contains about 12 g of hyaluronic acid. Although the highest concentrations of hyaluronic acid are found in connective tissues, most

hyaluronic acid, 56% (7 g), is found in the skin. The normal state of hyaluronic acid in tissues is as a free polymer. However, in some tissues such as the cartilage and tendons hyaluronic acid is bound to large glycoprotein structures (proteoglycans) or in other tissues to specific cell receptors (e.g. CD 44).

Tissue Hyaluronic acid mg/l

Synovial fluid 3500

Vitreous 200

Oocyte cumulus 500

Extracellular space mg/kg mg/kg

Cartilage 1200

Skin 200

Lung 150

Other mg/l

Serum 0.05

Intracellular Absent

All tissue 200

Table 6. Hyaluronic acid concentration in different tissues.

Metabolism

The metabolism – the biosynthesis and the catabolism – of hyaluronic acid is in many ways unique. The biosynthesis occurs via an enzyme complex within the cellular membrane, and the removal and degradation of hyaluronic acid is mediated by receptor binding followed by intracellular degradation. This process is very fast and efficient.

The enzyme complex producing hyaluronic acid is not situated within the cell but is maintained within the cell membrane. The two basic sugar units are added onto the growing hyaluronic acid chain from the cell interior, and the hyaluronic acid product is released directly into the surrounding extra cellular medium. Many different cells have the capacity to produce hyaluronic acid, e.g. fibroblasts, synovial cells, endothelial cells, smooth muscle cells, adventitial cells, and oocytes.

The overall turnover rate of hyaluronic acid is fast compared to other extra cellular components such as collagen. The half-life of hyaluronic acid in most tissues ranges from 0.5 to a few days. In skin the half-life is <24 hours. The daily turnover of

hyaluronan is in the order of one-third of the total body content, a rate similar to that of albumin. In a normal human body, about 3 grams of hyaluronic acid is catabolized each day.

The very fast turnover rate of hyaluronic acid takes place in a series of steps as outlined as follows. First, the large hyaluronic acid molecules move at a remarkable speed by means of a reptation (reptile movement) mechanism. The flexible

molecules disentangle and move out of the molecular network with a snake-like motion. Cell receptors bind the free hyaluronic acid molecules, which are engulfed by the cells. Intracellular enzymes in lysosomes subsequently degrade the hyaluronic acid to its basic constituents.

Physiological function

Hyaluronic acid is an important component of the extra cellular space, helping to maintain proper structure and function of tissues by:

• Creating volume

• Lubricating tissues

• Affecting cell integrity, mobility and proliferation

The physiological function of hyaluronic acid is based on the large volume and viscosity of is hydrophilic molecular network. In the extracellular space, the hyaluronic acid network holds large amounts of water. Elevated levels of extra cellular

hyaluronic acid accompany processes that require cell movement and tissue

reorganization; when cells need space for motility and separation these functions are performed in a hyaluronic acid medium. The hyaluronic acid network assists in cell differentiation, cell migration, tissue morphogenesis, embryogenesis, and wound repair. Moving tissues are lubricated by hyaluronic acid. Such effects are dependent on the rheological status of the fluid which is largely a function of the hyaluronic acid molecular weight. The high viscosity and elasticity of hyaluronic acid solutions creates thick layers of unstirred fluid that protect the tissues under movement.

Figure 18. Disappearance of hyaluronic acid from skin.

Biocompatibility

In general, biomolecules synthesized by different species differ in chemical composition. The difference in amino acid composition of proteins and sugar

components of glycoproteins often makes these molecules antigenic to other species or even other individuals of the same species. In contrast to other biomolecules, hyaluronic acid is independent of source as the chemical structure is invariant. This also applies to the hyaluronic acid produced by bacteria. The bacteria with a

protective coat of hyaluronic acid are not as easily recognized as foreign by the immune system and the inflammatory reaction will be greatly reduced.

NASHA: NON-ANIMAL STABILIZED HYALURONIC ACID⁹⁹

The development of NASHA was based on three basic considerations:

• Pure hyaluronic acid.

• Synthesis to obtain the desired physical form and sufficient residence time in the various clinical applications.

• Safety, no side effects, so that this synthetic hyaluronic acid is virtually identical to native hyaluronic acid without contaminants.

Starting material

The hyaluronic acid used in the manufacture of NASHA is biosynthesized from a proprietary non-animal source. The molecular weight is ~1 million. Higher molecular weights are not needed as the hyaluronic acid is “stabilized”, polymerized in a particular viscous form.

The raw material for NASHA is manufactured with a very high order of product purity though a proprietary process from non-animal cells. The presence of potentially harmful components such as viruses, proteins and endotoxins, those of animal origin in particular, is excluded.

Stabilization and manufacturing

The manufacturing of NASHA is by Q-Med AB, Uppsala, Sweden and includes stabilization of hyaluronic acid to form NASHA, polymerization into a continuous 3- dimensional molecular network by a proprietary process that maintains the ultimate tolerance of native hyaluronic acid; in this way a gel of any shape and form can be produced. Gel particles of defined sizes are produced depending on the intended application so that a defined polymer is produced with a physical form that suits the intended use, improves shelf life, and produces appropriate the residence time

following injection from a few days to many months depending on the specific degree of polymerization.

The products are steam-sterilized to a high sterility assurance level so that the probability of finding a 5 ml syringe of NASHA gel containing a microorganism is less than 1 in 1 million.

The modification needed to obtain the stabilized NASHA is presented in figures 19 and 20:

Figure 19. A hyaluronic acid molecule with the flexible molecular network entangles with its neighbors. This entanglement strongly hampers the movement of the molecules sideways.

However, individual molecules are capable of moving within the flexible molecular network at a remarkable speed by means of a snake-like movement called reptation.

Figure 20. In the NASHA products, the hyaluronic acid molecules are stabilized to a minor degree. The stabilization is accomplished using a compound that does not cause any significant biological reaction. Due to the high molecular weight of the starting material (1 million) minute amounts of stabilization are needed to obtain a few permanent linkages that

join all the hyaluronic acid molecules in the solution, thus forming a continuous gel.

Therefore, very low amounts of stabilizer are needed.

Biocompatibility

The biocompatibility of NASHA has been extensively tested both in vitro and in vivo following the guidelines in the ISO (International Organization for

Standardization) 10993 standard on “Biological Evaluation of Medical Devices”.

These biocompatibility tests have demonstrated that NASHA:

• is neither cytotoxic nor genotoxic

• does not give rise to any acute, subacute, or chronic effects

• does not give rise to any hypersensitivity reactions Residence time of NASHA

The turnover of endogenous hyaluronic acid is very fast and efficient. In most tissues the half-life varies from half a day to a few days so that exogenous hyaluronic acid implanted into a tissue will disappear within this short time. The residence time may be slightly modified by changing the molecular size or concentration of

hyaluronic acid, or by the modifying the method of application. However, modification of the hyaluronic acid molecular weight will increase the residence time only slightly, by a factor <2.

In healthy tissue, the extracellular degradation of NASHA is by free radicals, present in very low concentrations in normal tissue resulting in very slow degradation of NASHA with slow release of free hyaluronic acid chains, which are catabolised by the same mechanism as the endogenous hyaluronic acid described above. As a consequence of the mild stabilization in NASHA, the residence time in e.g. the skin has been increased from a few days to many months, sometimes almost up to a year^100-102.

The residence time of non-animal stabilized hyaluronic acid, NASHA, is

dependent on the size of the gel particles, the concentration of stabilized hyaluronic acid, and the existence of inflammatory reactions in the area. The smaller the gel particle size, the easier it will escape from its place of residence. Too large gel particles will rupture the tissue and cause reparative inflammatory reactions that will speed up the degradation. Hence a careful match of gel size to tissue is essential.

Gels of any shape and form

The NASHA gel can be manufactured to almost any shape and form. Depending on the clinical demand, the gels can be thick or thin, dense or loose as well as

containing big or small gel particles. For the purpose of tissue augmentation, the size of the gel particles has to match the density of the tissue. For facial augmentation e.g. several products with different size of the gel particles have been developed.

Clinical use of NASHA

NASHA™ has been successfully used for treatment of knee and hip osteoarthritis^103-105or facial tissue augmentation^106-108, for treatment of vesico-ureteral reflux in children^109,110, and for treatment of urinary stress incontinence in women^111-

113.

PART II (Specific study)