Ординатура / Офтальмология / Английские материалы / Ocular Neuroprotection_Levin, Polo _2003

stomach and the fluid is administered slowly (Fig. 2). With practice, an experienced technician can feed several hundred animals in a day using this method. The primary complication with gavage is entering the needle down the trachea and into the lungs. If this occurs the needle may become obstructed and the animal may begin to cough or struggle vigorously after the fluid is introduced. Fluid may also come out of the nose. At this point the needle should be withdrawn immediately. If it appears that fluid has been injected into the lungs, the animal should be euthanized. Another complication is the rupture of the esophagus or trachea due to excessive force when entering the needle. The resulting tissue damage can lead to hydrothorax after fluid application. The gavage method was

Figure 2 Diagram showing the gavage method for drug delivery in mice. (From Ref. 21.)

used successfully to introduce FK506 into rats to test its neuroprotective effects on retinal ganglion cells after optic nerve crush [22].

The advantages of the gavage method is greater control over the dose and the timing of application of a target drug. The drawback with gavage feeding is that it is more labor-intensive for the researcher and disruptive to the animals, although they will become accustomed to repeated feedings by this method.

B. Intraperitoneal Delivery

Intraperitoneal injections can be a very efficient method of systemic drug delivery [21]. Injections are made into the caudal left abdominal quadrant to avoid the cecum in the right. The animal should be held dorsally with the head and tilted away and down from the holder (Fig. 3). The needle should be introduced with a quick firm motion to ensure passage through the abdominal musculature. After

Figure 3 Diagram showing intraperitoneal injections into the mouse. (From Ref. 21.)

Figure 4 Diagram of subcutaneous injections into the mouse. (From Ref. 21.)

penetration, pull back on the syringe plunger. The presence of any fluid is indicative of penetration of an abdominal organ and the needle should be repositioned. Depending on the compound, injection into an abdominal organ should have no serious consequences other than altering the rate of absorption of the drug. Up to 1 mL of fluid can be introduced by this method into a mouse, but between 100 and 400 L is typical. This method has been used successfully to test the neuroprotective effects of brimonidine on retinal ganglion cells [23,24].

An alternative to intraperitoneal injections is subcutaneous administration of drug (Fig. 4). In general, access of an injected compound to the bloodstream is less rapid than with intraperitoneal injections, but this is usually not an issue with experiments that require prolonged drug exposure. In addition, subcutaneous injections are less likely to damage the animal, and therefore may be more desirable for conditions that require repeated dosing.

C.Intravenous Delivery

Drugs introduced intravenously (I.V.) can reach the retina very rapidly. In humans, for example, fluorescein injected into a vein of the arm reaches the retinal arteriole system in 12–15 s and completely fills the vasculature of the retina and choroid within 25 s in a healthy person. The best veins for delivering drugs vary with the animal used. The most accessible site for an I.V. injection in the mouse and rat is in one of the lateral tail veins, which are easily found although quite small [21]. Injections are typically carried out by placing the mouse in a restraining device with a hole that allows the tail to stick out (Fig. 5). Warming the tail will dilate the vein for better detection. Injections are made with a 27-G

Figure 5 Diagram of intravenous injection into a mouse through the lateral tail vein. The mouse is constrained in a chamber with the tail extending through a hole. (From Ref. 21.)

needle (or smaller), which is inserted into the vein and extended 1–2 mm. A lack of resistance to the injected material is indicative of successful entry into the vessel. In some cases, it may be possible to see blanching of the vein as the injected fluid fills it. The major drawback with this method of delivery is the inability to successfully perform repeated injections on some animals (such as mice) because of scarring of the veins.

ACKNOWLEDGMENTS

This work was supported in part by NIH grant R29 EY 12223, a grant from the American Health Assistance Foundation, and by an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology at the University of Wisconsin. The authors are grateful for the helpful discussions of Drs. Alyson Jarvis, T. Michael Nork, and Barbara Faha, and the assistance of Mr. Lou Kohl.

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II. NEUROPROTECTIVE STRATEGIES

A variety of gene delivery strategies have been explored to promote neuronal survival in the retina following injury or disease. A gene transfer protocol that leads to the complete and permanent rescue of degenerating retinal neurons is yet to be established. However, the following strategies successfully enhanced cell survival by delaying, in some cases considerably, the time-course of neurodegeneration.

A.Gene Supplementation

Many retinal disorders are caused by genetic defects that result in a lack of one or more essential gene products. Delivery of a healthy copy of the mutated gene to the affected cell may restore loss-of-function deficits and lead to neuroprotection. This approach has been tested in the retinal degeneration (rd) mouse, a model of autosomal recessive human retinitis pigmentosa (RP). Defects in the rod specific cGMP phosphodiesterase β-subunit (β-PDE) underlie irreversible and rapid photoreceptor loss in this model [1,2]. Transient rescue of the rd phenotype was achieved following delivery of the wild-type β-PDE cDNA into the retina using several viral vectors; adenovirus (Ad) [3] encapsidated Ad (gutted vector) [4], adeno-associated virus (AAV) [5], and lentivirus (LV) [6]. Furthermore, Ali et al. demonstrated that supplementation of the peripherin-2 gene using AAV vectors resulted in the preservation of outer segment ultrastructure and function for up to 10 months in the rds mouse [7], a model for autosomal dominant RP and macular dystrophy.

B.Ribozyme Therapy

Retinal diseases caused by dominantly inherited mutations may result in the production of abnormal gene products that affect cellular trafficking, metabolism, and function. Cell death due to accumulation of these toxic products can be minimized by delivery of ribozymes, small RNA molecules that can be designed to cleave mutant RNA transcripts while leaving the wild-type mRNAs intact. Lewin et al. demonstrated morphological and functional protection of photoreceptors following AAV-delivery of ribozymes in a mutant rhodopsin transgenic rat model of autosomal dominant RP [8]. Long-term studies on the effectiveness of this form of therapy indicated that the progression of photoreceptor loss was considerably delayed for up to 8 months of age [9].

C.Neurotrophic Factor Therapy

The main limitation of the gene correction therapies mentioned above is that the genetic basis of prevalent forms of retinal disorders, such as glaucoma or age-

related macular degeneration, remains unknown. In addition, retinal diseases that arise from mutations in several genes (polygenic diseases) or a combination of environmental and genetic factors represent a challenge in the design of potential gene therapies. Alternative therapies would involve the use of neurotrophic factors, with a broad spectrum of action, capable of providing a more generic form of neuroprotection. Neurotrophic factors bind to specific cell surface receptors triggering intracellular signaling pathways that lead to neuronal survival [10– 12]. Because most neurotrophic factors are secreted and diffuse well within the retina, this type of approach allows one to select either the affected cell or its supporting cells as targets for gene transfer. For example, delivery of the brainderived neurotrophic factor (BDNF) gene to Mu¨ller glial cells using an Ad vector resulted in temporary protection of retinal ganglion cells in an optic nerve axotomy rat model [13]. Ad-mediated delivery of ciliary neurotrophic factor (CNTF) has been shown to slow photoreceptor loss in the rd and rds mouse [14,15]. Basic fibroblast growth factor (bFGF) delivered by Ad vectors delayed retinal degeneration in the Royal College of Surgeons (RCS) rat [16]. More recently, AAV-mediated delivery of bFGF delayed photoreceptor cell death in transgenic rats carrying a mutant rhodopsin [17] and retinal ganglion cell loss after axotomy [18].

D. Inhibition of Apoptosis

Apoptosis or programmed cell death appears to be a common mechanism of neuronal loss in the injured or degenerating retina. Retinal ganglion cells have been shown to die by apoptosis after optic nerve axotomy and in experimental glaucoma [19–21]. Photoreceptors also die by apoptosis in several inherited mouse models of RP [22,23], the RCS rat [24], light-induced retinal damage [25] and experimental retinal detachment in the rat [26]. The identification of intracellular components of the cell death program (e.g., bcl-2 family members and caspases) has motivated experimental strategies involving transfer of antiapoptotic genes. For example, gene delivery of bcl-2 using Ad resulted in moderate protection of photoreceptors in the rd mouse model [27]. Recently, Ad-medi- ated gene transfer of the X-linked inhibitor of apoptosis protein (XIAP) resulted in partial protection of retinal ganglion cells following axotomy [28] and high intraocular pressure [29].

III. SELECTION OF A GENE DELIVERY SYSTEM

Genes can be introduced into cells via nonviral and viral vectors. Nonviral methods include liposome-mediated DNA transfer, DNA carried on ballistic metal particles (“gene gun”), and micro-injection techniques [30]. These gene transfer