June 2006
Volume 47, Issue 6
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Physiology and Pharmacology  |   June 2006
Selective Cell Uptake of Modified Tat Peptide–Fluorophore Conjugates in Rat Retina in Ex Vivo and In Vivo Models
Author Affiliations
  • Edward M. Barnett
    From the Departments of Ophthalmology and Visual Sciences and
  • Boobalan Elangovan
    From the Departments of Ophthalmology and Visual Sciences and
  • Kristin E. Bullok
    Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
  • David Piwnica-Worms
    Molecular Biology and Pharmacology, and the
    Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
Investigative Ophthalmology & Visual Science June 2006, Vol.47, 2589-2595. doi:10.1167/iovs.05-1470
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      Edward M. Barnett, Boobalan Elangovan, Kristin E. Bullok, David Piwnica-Worms; Selective Cell Uptake of Modified Tat Peptide–Fluorophore Conjugates in Rat Retina in Ex Vivo and In Vivo Models. Invest. Ophthalmol. Vis. Sci. 2006;47(6):2589-2595. doi: 10.1167/iovs.05-1470.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To determine the pattern of retinal uptake of modified Tat peptide–fluorophore conjugates in the rat after ex vivo application and intravitreal injection.

methods. Modified Tat peptide (RKKRRORRRGC) was conjugated at the C terminus to Alexa Fluor 594 to enable visualization of uptake. In the ex vivo model, posterior segments were incubated for up to 120 minutes in peptide solution. In the in vivo model, intravitreal injections of 5 μL peptide solution were performed in anesthetized rats, which were then euthanatized from 1 hour to 7 days after injection. Retinal and optic nerve paraffin sections were examined for fluorescent labeling. Immunohistochemistry for retinal cell markers was performed to identify cell types exhibiting uptake.

results. The pattern of labeling seen in retinal sections was highly similar for the ex vivo and in vivo experiments, with specific uptake by retinal ganglion cells (RGCs) and by a subset of inner nuclear layer cells. The pattern of labeling remained specific even at the later time points. In the in vivo model, fluorescence was also noted in the nerve fiber layer and anterior optic nerve, extending posteriorly along the optic nerve at later time points.

conclusions. A specific pattern of uptake for modified Tat peptides was consistently seen in the rodent retina. Given the preferential uptake of these peptides by RGCs and the potential to conjugate diverse moieties, modified Tat peptides may be useful for delivery of therapeutic agents or molecular imaging probes to RGCs.

In the retina, as in other tissues, effective delivery of charged molecules or high molecular–weight compounds into living cells is limited by the plasma membrane. Most peptide-based drugs and imaging probes exhibit poor cellular uptake, limiting their usefulness. One approach has been the delivery of genes to cells by means of viral (active) or nonviral (passive) vectors with subsequent expression. This approach has been limited by a number of issues including toxicity, immunogenicity, efficiency of uptake, and specificity of expression. 1  
Polypeptide sequences of positively charged amino acids have been shown to enhance the uptake of impermeant molecules into cells. 2 These peptides have been referred to in the literature as protein transduction domains, cell penetrating peptides, or permeation peptides. 2 3 4 The exact mechanism by which these peptides are taken up into cells remains under debate, but a number of studies suggest macropinocytosis and related endocytic pathways may contribute. 5 6 7 8 9 10 Nonetheless, permeation peptides are useful for translocating substrates such as proteins, peptides, nucleic acids, or imaging probes into cells in vivo. 11 12 13 14 One such peptide sequence, derived from the HIV-1 Tat protein, has been studied extensively. 15 16 17  
The eye is an attractive model in which to study permeation peptide–mediated uptake given its accessibility and the relative ease and specificity of intraocular delivery. The delivery of therapeutics or molecular imaging probes to the retina is of particular interest in that retinal neurons are affected by a number of acquired and inherited degenerative conditions. Inner retinal neurons, such as the retinal ganglion cells (RGCs), which degenerate in glaucoma, are particularly accessible through an intravitreal approach. In this study, we examined the retinal uptake of modified Tat peptides linked to a fluorophore after ex vivo application and in vivo intravitreal injection in the rat. 
Methods
Modified Tat Peptide Conjugates
The permeation peptide sequence used was based on the wild-type HIV-1 Tat sequence (Tat47–60). D-isomer amino acid peptides were used to prevent in vivo proteolysis. The D-isomer has also been shown to have enhanced cellular uptake. 4 Substitution of ornithine for glutamine was also found to increase cellular uptake. 4 A second peptide sequence from a viral protein (HSV-1 VP-22) served as a control. Both peptides were conjugated at the C terminus to a fluorescent marker for direct visualization of cellular uptake within retinal sections. 
The peptides Ac-rkkrrorrrgc-NH2 (modified Tat) and Ac-plssifsrigdp-AHA-εkgc-NH2 (control) were prepared by solid-phase peptide synthesis (Tufts University Peptide Synthesis Core Facility, Boston, MA) using standard BOP/HOBt coupling chemistry and all D N-α-Fmoc-protected amino acids. 18 Peptides were obtained pure and were conjugated to Alexa Fluor 594 maleamide (AF; 1.2 equiv; Molecular Probes, Eugene, OR) by thiol conjugation at ambient temperature in 0.5 × PBS for 2 hours. Quantitative yields were observed for all reactions, as analyzed by C18 reverse-phase HPLC (RP-HPLC). Peptides were purified by RP-HPLC at a flow rate of 1 mL/min using, as eluent, Solvent A (0.1% trifluoroacetic acid in 5% acetonitrile/95% water [0.1% TFA/(5% CH3CN/H2O)]) modified with Solvent B (0.1% trifluoroacetic acid in 90% acetonitrile/10% water [0.1% TFA/(90% CH3CN/H2O)]) by a linear gradient of 100% A to 40% A over 40 minutes (modified Tat) or 100% A to 65% A over 15 minutes to 50% A at 30 minutes (control) before washing with Solvent B to obtain the following pure peptides: Ac-rkkrrorrrgc(AF)-NH2 (modified Tat, t R = 21.6 minutes; m/z: 2414.0; calc: 2412.7) and Ac-plssifsrigdp-AHA-εkgc(AF)-NH2 (control, t R = 25.9 minutes; m/z: 2618.0; calc: 2617). According to previous studies, isolated doublets from the modified Tat peptides were determined by electrospray mass spectrometry (ESMS) to have identical mass, indicating that the doublet represents two independent conformers of the desired product. 19  
As a control for uptake mediated by the fluorophore, nonreactive Alexa Fluor 594 was obtained through succinimidyl ester hydrolysis by incubating Alexa Fluor 594 succinimide (Invitrogen, Carlsbad, CA) in water at pH 9 for 9 hours at ambient temperature. RP-HPLC analysis using the above gradient for purification of the modified Tat peptides revealed an increase over time of a doublet peak at earlier retention times (t R = 21.1/22.2) than the parental doublet peak (t R = 23.4/24.1). Near completion of hydrolysis (80%) was observed as determined by integration of the resultant doublet peak. The hydrolyzed doublet peaks were isolated, analyzed by ESMS, and determined to have identical mass (m/z: 723.3; calc. 722.9), indicating that the doublet represented two independent conformers. 
Animals
Male Brown Norway rats weighing 200 to 300 g each were purchased from Charles River Laboratories (Wilmington, MA). All animal procedures in this study were approved by the Animal Studies Committee at Washington University in St. Louis School of Medicine and were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Ex Vivo Application
For the ex vivo model, male Brown Norway rats were euthanatized through intraperitoneal injection of 150 mg/kg sodium pentobarbital, and the eyes were enucleated. Eyes were washed with PBS, and corneas and lenses were removed. The posterior segments were incubated in either 20-μM or 40-μM concentrations of modified Tat peptide or control peptide conjugates in PBS (pH 7.4) for 20, 40, or 120 minutes at room temperature in a covered cell culture dish in room air. Posterior segments were rinsed with PBS for 5 minutes three times and then were placed in 4% paraformaldehyde for 1 hour at room temperature. All time points were examined in triplicate for both concentrations. 
Extraocular Injections
To determine whether the modified Tat peptide conjugates would penetrate the eye after extraocular injection, 10 μL of 1 mM peptide solution was injected into the subconjunctival space in a single eye. In addition, intraperitoneal (1 mL of 1 mM solution) and intravascular (500 μL of 1 mM solution through the tail vein) injections were performed to determine whether the modified Tat peptide conjugates would cross the blood-retinal barrier (BRB) and the blood-aqueous barrier. Animals were euthanatized 2 days after injection, and the eyes were enucleated. 
In Vivo Intravitreal Injections
To confirm our ex vivo results using an in vivo model, intravitreal injections were performed as follows. Rats were anesthetized by intraperitoneal injection of 1 mL/kg solution of a cocktail containing 1 mL ketamine (100 mg/mL) and 0.15 mL xylazine (100 mg/mL), and the pupil was dilated with 1% tropicamide drops. Micropipettes were created from glass pipettes pulled on a pipette puller and beveled using a Dremel drill with a sander attachment. Injections were performed using micropipettes threaded into polypropylene tubing attached to a 250-μL Hamilton syringe with a 5-μL autoinjector. The syringe and tubing were filled with mineral oil to reduce dead space. The micropipette penetrated the sclera at 2 mm posterior to the limbus temporally, and 5 μL of 250 μM modified Tat peptide–Alexa Fluor 594 conjugate in PBS solution was injected into the vitreous through the pars plana. The success of the injection was verified by direct visualization of a red reflex through the dilated pupil. At 1 hour, 4 hours, 24 hours, and 7 days after injection, rats were euthanatized with lethal doses of sodium pentobarbital (150 mg/kg) and were perfusion fixed with ice-cold 4% paraformaldehyde. The globes were then enucleated with a portion of the anterior optic nerve. Procedures at all time points were performed in triplicate. Injections were performed in similar fashion and in triplicate using the control peptide or hydrolyzed Alexa Fluor 594, with euthanasia and enucleation at 1 day after injection only. 
To determine whether transport of modified Tat peptide–fluorophore conjugates occurred along RGC axons, prechiasmatic optic nerves were excised after euthanasia and fixation perfusion after unilateral intravitreal injection of 10 μL of 1.0 mM peptide in PBS solution. The larger volume and higher concentration were used to enhance the detection of signal present in the optic nerves. 
Tissue Processing
Posterior segments, globes with the attached anterior optic nerves, or prechiasmatic optic nerves were placed in 4% paraformaldehyde at room temperature until paraffin embedding. Ocular and optic nerve paraffin blocks were cut into 4-μm sections and were stained with DAPI or 2-Pro-3 iodide (Invitrogen, Carlsbad, CA). 
Immunohistochemistry
To identify the cell types in the inner retina that were positive for modified Tat peptide–fluorophore uptake, paraffin-embedded ocular sections were immunolabeled with antibodies to several retinal neuronal markers using standard protocols. Briefly, ocular sections were deparaffinized, rehydrated, and blocked with 10% normal donkey serum. Blocked sections were incubated overnight at 4°C with primary antibodies in a humidified chamber. Slides were washed three times with TBST (Tris-buffered saline with 0.1% Tween 20) followed by incubation with Alexa Flour 488–conjugated secondary antibodies for 30 minutes at room temperature. Slides were washed three times for 10 minutes with TBST and were mounted with coverslips for microscopic evaluation. Sections treated only with secondary antibody were used as controls for background labeling. Primary antibodies used included mouse monoclonal anti–Brn-3a (1:50), mouse monoclonal anti-NF70 (1:200), rabbit polyclonal anti-Calbindin D-28 (1:1000), mouse monoclonal anti–glutamine synthetase (1:1000), goat polyclonal anti-Calretinin (1:1000; Chemicon, Temecula, CA), and mouse monoclonal anti–protein kinase (1:100; Sigma, St. Louis, MO). 
Results
Ex Vivo Application
The pattern of fluorescent staining in the retina after ex vivo application was consistent across the three time points and the two concentrations examined. Prominent labeling of cells in the RGC layer was noted (Fig. 1) . Uptake by a subset of anterior inner nuclear layer (INL) cells was also a consistent finding throughout the retina (Fig. 1) . No fluorescence was seen in the outer retina. A cytoplasmic pattern of fluorescent labeling predominated, but there was no labeling in axons or dendrites. 
Intravitreal Injection
The pattern of fluorescent staining in the retina after intravitreal injection in live rats was highly similar across the time points examined (Fig. 2) . Fluorescence was again noted in the RGC layer, now with prominent nuclear labeling. In contrast to the labeling seen after ex vivo application, strong fluorescence was seen in RGC axons in the nerve fiber layer (NFL) even at the earliest time point examined (1 hour after injection) (Fig. 2A) . A dendritic pattern of labeling also extended into the inner plexiform layer (IPL). Overall, there was a gradual decrease in RGC layer fluorescence with increasing time after injection. 
As seen with ex vivo application, fluorescent labeling was typically noted in a subset of anteriorly located cells in the INL. This was either faint or nonexistent at 1 hour after injection and became more prominent 1 day after injection (Fig. 2) . The pattern of uptake by cells in the INL remained fairly specific even at 7 days after injection, though fluorescence was noted in a larger number of INL cells and in occasional outer nuclear layer cells. There was no evidence of retinal damage or violation of the lens capsule caused by intravitreal injection in any ocular section. 
Immunohistochemistry
After immunolabeling with antibody against Brn-3a, cells in the RGC layer demonstrating uptake of modified Tat peptide–fluorophore conjugates were double labeled (Fig. 3) , confirming the identity of these cells as RGCs. Calretinin immunolabeling resulted in double labeling of occasional cells in the INL, consistent with modified Tat peptide–fluorophore conjugate uptake by a subset of amacrine cells in the INL. However, Alexa Fluor 594–positive cells in the INL were not double labeled after immunolabeling for calbindin D-28, protein kinase C, or glutamine synthetase, indicating that they were not horizontal, bipolar, or Müller cells (data not shown). 
Control and Extraocular Injections
No uptake was seen in the retina after intraocular injection of the control peptide–fluorophore conjugate or with nonreactive Alexa Fluor 594 (Fig. 4) . In addition, no retinal uptake was noted after subconjunctival, intraperitoneal, or intravascular injections of modified Tat peptide–fluorophore conjugates (data not shown). 
NFL and Optic Nerve Labeling
Labeling of nerve fibers (axons of RGCs) in the NFL was noted 1 hour after intravitreal injections and decreased with increasing duration after injection (Fig. 2A 5) . One hour after injection, labeling was noted in the nerve fiber layer of the optic nerve head. Four hours after injection, fluorescence was noted deep in the anterior optic nerve. The pattern of labeling, compared with immunohistochemistry for NF-70, was consistent with fluorescence in RGC axons in the optic nerve head (Fig. 5) . By 1 day after injection, this pattern of fluorescence extended further into the optic nerve head (Fig. 5)
To examine whether fluorescent labeling spread further into the optic nerve, the prechiasmatic optic nerve was isolated and examined in coronal sections 7 days after injection. After unilateral intravitreal injections, sections of the optic nerves revealed ipsilateral fluorescence (Fig. 6) . Although fluorescence was noted posteriorly to the level of the optic chiasm, a gradual diminution of signal was seen with increasing distance from the globe. 
Discussion
It is well established that a peptide sequence derived from the HIV-1 Tat protein, consisting of a cluster of basic amino acids, improves cellular delivery of attached molecules. 5 20 This property has already been used to enhance the cellular uptake of a number of biologically active molecules in several different tissues. 18 19 21 22 23 Despite these results, the exact mechanism of cellular entry used by the Tat peptide remains controversial, with evidence for the involvement of endocytic and non-endocytic pathways. 5 6 7 8 9 24 The literature shows no evidence of a cell surface Tat receptor. Contradictory results across experimental models using Tat peptides likely occurs because of a number of factors, including differences in the Tat peptide sequence used, the wide variety of cell lines and tissues studied, and the diversity of cargoes attached to Tat peptides. 5  
Differences across studies in the method of tissue fixation are another possible cause for disparate results. Because of their positively charged nature, Tat peptides are expected to attach to the negatively charged plasma membrane. Adsorption of Tat peptide conjugates to cellular membranes, with little or no transduction into the cell, has been demonstrated. 25 26 The use of certain fixatives, such as alcohols or acetone, may result in an overestimation of transduction by enabling adherent Tat peptide constructs to enter the cell through permeabilized cell membranes. Given that the endocytic pathway has been implicated in Tat peptide uptake, fixatives might also have an effect on the cellular distribution of labeling due to alteration of internal cellular membranes. 27 Artifactual evidence of Tat peptide–mediated transduction into the cytoplasm and nucleus has been observed after methanol and acetone fixation. 7 Consistent with this, nonspecific cytoplasmic labeling in all nuclear layers of the retina was noted when tissue was fixed with graded alcohols only (data not shown). 
Our results are consistent with true rather than artifactual uptake of the modified Tat peptide–fluorophore conjugate used in this study. The pattern of retinal uptake was consistent across the ex vivo and in vivo models. Although the intracellular pattern of labeling in RGCs differed across the two models, this might have reflected cellular changes in the ex vivo model. The pattern of uptake was also consistent across concentration and time after injection. This specificity argues against a fixation-induced artifact, which would have resulted in a more diffuse, nonspecific pattern of labeling. No uptake was noted of nonreactive Alexa Fluor 594 or of a control peptide sequence derived from a viral protein. Most important, the spread of fluorescent labeling into the optic nerve along RGC axons, as seen in the in vivo model, would not be expected to occur in the absence of true intracellular uptake by RGCs. 
Little is known regarding what happens to the linkage between the Tat peptide and its cargo on entry into cells, though this is likely influenced by the tissue or cell type, the cargo, and the chemical nature of the linkage. 5 It is not possible to say, based on our results, whether the modified Tat peptide conjugate remained intact after RGC uptake or whether labeling in the NFL and optic nerve represented transport of the cleaved fluorophore only. Given the lengths of their axons, RGCs would be an excellent model in which to study this issue. It is possible that labeling of cells in the INL represented neuron-to-neuron spread, particularly given the dendritic labeling noted in the inner plexiform layer, but no convincing evidence was observed of transsynaptic spread of these modified Tat peptide conjugates. It is interesting, however, that transsynaptic spread of the HIV-1 Tat protein was demonstrated in the rodent brain. 28  
The spread of fluorescence to the optic nerve suggests that at least the cargo, in this case Alexa Fluor 594 and possibly the entire Tat peptide conjugate itself, may enter the axonal transport pathway in RGCs. Although labeling was often noted in the retinal NFL even at 1 hour after injection, the earliest time point examined, the temporal pattern of spread into the anterior optic nerve head and along the optic nerve is most consistent with uptake by RGC bodies with eventual axonal transport. It remains possible that retinal NFL labeling reflects direct uptake by RGC axons in this layer, but the lack of NFL labeling in the ex vivo model suggests that this in vivo finding reflects cell body uptake with later transport. In addition, in vitro retinal cell culture studies of cellular uptake of these modified Tat peptides (data not shown) and the ex vivo model show that cellular uptake occurs within 20 minutes of exposure. Thus, transport within cells during the time it takes to fix tissue is possible, even with nearly immediate fixation-perfusion. The gradual decrease in fluorescence of cell bodies in the RGC layer and axons in the NFL from 1 hour to 7 days after injection is also consistent with the spread of the fluorophore from RGC bodies into and along RGC axons. 
The selective uptake of Tat peptide–fluorophore conjugates by RGCs and cells in the INL persisted with only minor variation, even at time points ranging up to 1 week after injection. This is in contrast to earlier in vitro and in vivo studies of Tat peptides that suggested uptake would occur in almost any cell type. 29 30 Indeed, this lack of specificity was considered a drawback to the use of Tat peptides as a targeting moiety to deliver biologically active compounds to cells. 20 More recent evidence suggests that there are a number of cell types in which Tat peptide–mediated transduction either does not occur or occurs at low efficiency. 31  
Although previously published articles on Tat peptide–mediated transduction in the retina have assayed for the biologic activity of the cargo, this study has instead focused specifically on the temporal and topographic pattern of retinal uptake and intracellular spread of the Tat peptide conjugates. Dietz et al. 21 injected a Tat-Bcl-XL fusion protein intravitreally in rodents after optic nerve transection and were able to show reduced apoptosis. With the use of antibody directed against the recombinant protein, they were also able to demonstrate efficient transduction in the RGC layer but did not report uptake by other retinal layers. Harbor et al. 23 described intraocular injection of Tat peptide–based transducible peptides with oncoprotein inhibitors in a rabbit model of intraocular tumor but again focused on the biologic activity of the cargo. Using an approach similar to ours, Schorderet et al. 32 examined the pattern of retinal uptake of FITC-labeled D and L isomers of the wild-type Tat peptide sequence in the mouse. After intravitreal injection, they reported robust fluorescence in the RGC layer and the INL, with weaker labeling of the ONL. Fluorescence was noted in all three cellular retinal layers after subretinal injection. We did not perform subretinal injections in this study. Nonetheless, the relative absence of uptake by cells in the ONL in our study is noteworthy, particularly at time points as late as 7 days after injection. This is despite fluorescent labeling noted in the adjacent RPE layer, which suggests that this finding does not simply represent limited tissue diffusion of peptide solution to the outer retina. 
The lack of retinal uptake after intravenous and intraperitoneal injections of our modified Tat peptide constructs indicates that the blood-retinal and the blood-aqueous barriers effectively prevented their movement from the intravascular space into the eye. It cannot be assumed that this will be the case for all Tat peptide sequences or cargo. The published data on movement of various Tat peptide constructs across the blood-brain barrier, for example, shows that it is not always permeable for the uptake of Tat fusion proteins, 16 19 though evidence for uptake and biologic activity have been shown in several models. 30 33 34 The lack of retinal uptake after subconjunctival injection also suggests that the sclera acts as a barrier to the movement of the modified Tat peptide-conjugates into the eye. This is consistent with the lack of intraocular penetration demonstrated with a Tat peptide β-galactosidase construct after incubation of intact rat globes. 35 Although these findings indicate the necessity of direct intraocular delivery, they also suggest that modified Tat peptide conjugates so delivered will not exit the eye through vascular or transscleral routes. 
In conclusion, the rat retina showed a specific pattern of uptake after exposure to modified Tat peptide–fluorophore conjugates in ex vivo and in vivo models. Determining the basis for this specificity will require further study. In contrast, previous reports examining Tat peptide uptake by neurons in the brain and retina suggested a more nonspecific uptake by all cell types. The selective uptake by RGCs may prove useful because these cells are particularly attractive targets for molecular imaging and neuroprotective strategies given their degeneration in glaucoma. Although the uptake of permeation peptides can be expected to be dependent on the peptide sequence and on the cargo delivered, our results indicate that the selective delivery of cargo to RGCs may be possible using these modified Tat peptides. 
 
Figure 1.
 
Retinal uptake of modified Tat peptide–Alexa Fluor 594 conjugates after ex vivo application for 40 minutes. (A) Alexa Fluor 594. (B) Corresponding DAPI staining. Uptake of Tat peptide–fluorophore conjugates is seen in the RGC layer and a subset of INL cells. Original magnification, ×20.
Figure 1.
 
Retinal uptake of modified Tat peptide–Alexa Fluor 594 conjugates after ex vivo application for 40 minutes. (A) Alexa Fluor 594. (B) Corresponding DAPI staining. Uptake of Tat peptide–fluorophore conjugates is seen in the RGC layer and a subset of INL cells. Original magnification, ×20.
Figure 2.
 
In vivo retinal uptake of modified Tat peptide–Alexa Fluor 594 conjugates after intravitreal injection. (A) One hour after injection. (B) One day after injection. (C) Seven days after injection. (DF) Corresponding DAPI staining. A consistent pattern of fluorescent labeling is seen in the RGC layer and in a subset of anterior INL cells. Original magnification, ×20.
Figure 2.
 
In vivo retinal uptake of modified Tat peptide–Alexa Fluor 594 conjugates after intravitreal injection. (A) One hour after injection. (B) One day after injection. (C) Seven days after injection. (DF) Corresponding DAPI staining. A consistent pattern of fluorescent labeling is seen in the RGC layer and in a subset of anterior INL cells. Original magnification, ×20.
Figure 3.
 
Immunohistochemistry with Brn-3a after intravitreal injection of modified Tat peptide–Alexa Fluor 594 conjugates. (A) 2-Pro-3-iodide staining. (B) Brn-3a immunolabeling. (C) Modified Tat peptide–Alexa Fluor 594. (D) Merged image of panels (B) and (C). Cells in the RGC layer exhibiting uptake of modified Tat peptide–fluorophore conjugates are RGCs. Original magnification, ×40.
Figure 3.
 
Immunohistochemistry with Brn-3a after intravitreal injection of modified Tat peptide–Alexa Fluor 594 conjugates. (A) 2-Pro-3-iodide staining. (B) Brn-3a immunolabeling. (C) Modified Tat peptide–Alexa Fluor 594. (D) Merged image of panels (B) and (C). Cells in the RGC layer exhibiting uptake of modified Tat peptide–fluorophore conjugates are RGCs. Original magnification, ×40.
Figure 4.
 
Retinal uptake after intravitreal injections with control peptide–Alexa Fluor 594 or hydrolyzed Alexa Fluor 594 at 1 day after injection. (A) Control peptide (VP-22)–Alexa Fluor 594. (B) Hydrolyzed Alexa Fluor 594. (CD) Corresponding DAPI staining. No retinal uptake is detectable after the control injections. Original magnification, ×20.
Figure 4.
 
Retinal uptake after intravitreal injections with control peptide–Alexa Fluor 594 or hydrolyzed Alexa Fluor 594 at 1 day after injection. (A) Control peptide (VP-22)–Alexa Fluor 594. (B) Hydrolyzed Alexa Fluor 594. (CD) Corresponding DAPI staining. No retinal uptake is detectable after the control injections. Original magnification, ×20.
Figure 5.
 
Fluorescent labeling in the anterior optic nerve after intravitreal injection of modified Tat peptide–Alexa Fluor 594 (Tat-AF594). (A) One hour after injection, fluorescence was detected in the NFL but did not extend more deeply into the optic nerve. (B) Corresponding DAPI staining. (C) Four hours after injection, fluorescent labeling extended into the anterior optic nerve. (D) The same section immunolabeled for NF-70 to reveal RGC axons in the optic nerve. (E) Merged Alexa Fluor 594 (red) labeling and NF-70 (green) immunolabeling confirms that the former corresponds to the pattern of RGC axons. (F) One day after injection, fluorescent labeling extended further posteriorly in the optic nerve. Fluorescence is also noted in the retinal pigment epithelial layer. Original magnification, ×20.
Figure 5.
 
Fluorescent labeling in the anterior optic nerve after intravitreal injection of modified Tat peptide–Alexa Fluor 594 (Tat-AF594). (A) One hour after injection, fluorescence was detected in the NFL but did not extend more deeply into the optic nerve. (B) Corresponding DAPI staining. (C) Four hours after injection, fluorescent labeling extended into the anterior optic nerve. (D) The same section immunolabeled for NF-70 to reveal RGC axons in the optic nerve. (E) Merged Alexa Fluor 594 (red) labeling and NF-70 (green) immunolabeling confirms that the former corresponds to the pattern of RGC axons. (F) One day after injection, fluorescent labeling extended further posteriorly in the optic nerve. Fluorescence is also noted in the retinal pigment epithelial layer. Original magnification, ×20.
Figure 6.
 
Coronal optic nerve sections after unilateral intravitreal injection of modified Tat peptide–Alexa Fluor 594. (A) DAPI staining. (B) Alexa Fluor 594 fluorescent labeling. Prominent labeling is seen in the ipsilateral optic nerve only. Original magnification, ×10.
Figure 6.
 
Coronal optic nerve sections after unilateral intravitreal injection of modified Tat peptide–Alexa Fluor 594. (A) DAPI staining. (B) Alexa Fluor 594 fluorescent labeling. Prominent labeling is seen in the ipsilateral optic nerve only. Original magnification, ×10.
The authors thank Belinda McMahan and Zelma (Jean) Jones for their assistance in tissue processing. 
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Figure 1.
 
Retinal uptake of modified Tat peptide–Alexa Fluor 594 conjugates after ex vivo application for 40 minutes. (A) Alexa Fluor 594. (B) Corresponding DAPI staining. Uptake of Tat peptide–fluorophore conjugates is seen in the RGC layer and a subset of INL cells. Original magnification, ×20.
Figure 1.
 
Retinal uptake of modified Tat peptide–Alexa Fluor 594 conjugates after ex vivo application for 40 minutes. (A) Alexa Fluor 594. (B) Corresponding DAPI staining. Uptake of Tat peptide–fluorophore conjugates is seen in the RGC layer and a subset of INL cells. Original magnification, ×20.
Figure 2.
 
In vivo retinal uptake of modified Tat peptide–Alexa Fluor 594 conjugates after intravitreal injection. (A) One hour after injection. (B) One day after injection. (C) Seven days after injection. (DF) Corresponding DAPI staining. A consistent pattern of fluorescent labeling is seen in the RGC layer and in a subset of anterior INL cells. Original magnification, ×20.
Figure 2.
 
In vivo retinal uptake of modified Tat peptide–Alexa Fluor 594 conjugates after intravitreal injection. (A) One hour after injection. (B) One day after injection. (C) Seven days after injection. (DF) Corresponding DAPI staining. A consistent pattern of fluorescent labeling is seen in the RGC layer and in a subset of anterior INL cells. Original magnification, ×20.
Figure 3.
 
Immunohistochemistry with Brn-3a after intravitreal injection of modified Tat peptide–Alexa Fluor 594 conjugates. (A) 2-Pro-3-iodide staining. (B) Brn-3a immunolabeling. (C) Modified Tat peptide–Alexa Fluor 594. (D) Merged image of panels (B) and (C). Cells in the RGC layer exhibiting uptake of modified Tat peptide–fluorophore conjugates are RGCs. Original magnification, ×40.
Figure 3.
 
Immunohistochemistry with Brn-3a after intravitreal injection of modified Tat peptide–Alexa Fluor 594 conjugates. (A) 2-Pro-3-iodide staining. (B) Brn-3a immunolabeling. (C) Modified Tat peptide–Alexa Fluor 594. (D) Merged image of panels (B) and (C). Cells in the RGC layer exhibiting uptake of modified Tat peptide–fluorophore conjugates are RGCs. Original magnification, ×40.
Figure 4.
 
Retinal uptake after intravitreal injections with control peptide–Alexa Fluor 594 or hydrolyzed Alexa Fluor 594 at 1 day after injection. (A) Control peptide (VP-22)–Alexa Fluor 594. (B) Hydrolyzed Alexa Fluor 594. (CD) Corresponding DAPI staining. No retinal uptake is detectable after the control injections. Original magnification, ×20.
Figure 4.
 
Retinal uptake after intravitreal injections with control peptide–Alexa Fluor 594 or hydrolyzed Alexa Fluor 594 at 1 day after injection. (A) Control peptide (VP-22)–Alexa Fluor 594. (B) Hydrolyzed Alexa Fluor 594. (CD) Corresponding DAPI staining. No retinal uptake is detectable after the control injections. Original magnification, ×20.
Figure 5.
 
Fluorescent labeling in the anterior optic nerve after intravitreal injection of modified Tat peptide–Alexa Fluor 594 (Tat-AF594). (A) One hour after injection, fluorescence was detected in the NFL but did not extend more deeply into the optic nerve. (B) Corresponding DAPI staining. (C) Four hours after injection, fluorescent labeling extended into the anterior optic nerve. (D) The same section immunolabeled for NF-70 to reveal RGC axons in the optic nerve. (E) Merged Alexa Fluor 594 (red) labeling and NF-70 (green) immunolabeling confirms that the former corresponds to the pattern of RGC axons. (F) One day after injection, fluorescent labeling extended further posteriorly in the optic nerve. Fluorescence is also noted in the retinal pigment epithelial layer. Original magnification, ×20.
Figure 5.
 
Fluorescent labeling in the anterior optic nerve after intravitreal injection of modified Tat peptide–Alexa Fluor 594 (Tat-AF594). (A) One hour after injection, fluorescence was detected in the NFL but did not extend more deeply into the optic nerve. (B) Corresponding DAPI staining. (C) Four hours after injection, fluorescent labeling extended into the anterior optic nerve. (D) The same section immunolabeled for NF-70 to reveal RGC axons in the optic nerve. (E) Merged Alexa Fluor 594 (red) labeling and NF-70 (green) immunolabeling confirms that the former corresponds to the pattern of RGC axons. (F) One day after injection, fluorescent labeling extended further posteriorly in the optic nerve. Fluorescence is also noted in the retinal pigment epithelial layer. Original magnification, ×20.
Figure 6.
 
Coronal optic nerve sections after unilateral intravitreal injection of modified Tat peptide–Alexa Fluor 594. (A) DAPI staining. (B) Alexa Fluor 594 fluorescent labeling. Prominent labeling is seen in the ipsilateral optic nerve only. Original magnification, ×10.
Figure 6.
 
Coronal optic nerve sections after unilateral intravitreal injection of modified Tat peptide–Alexa Fluor 594. (A) DAPI staining. (B) Alexa Fluor 594 fluorescent labeling. Prominent labeling is seen in the ipsilateral optic nerve only. Original magnification, ×10.
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