RETINA: Immunohistochemical approaches to examine neuronal and glial responses to disease

Transcription

1 CL Davis meeting, Philadelphia, Dec 7, 2007 RETINA: Immunohistochemical approaches to examine neuronal and glial responses to disease William BELTRAN, DVM, PhD, Dipl. ECVO Assistant Professor of Ophthalmology School of Veterinary Medicine University of Pennsylvania Philadelphia, PA, USA Ph: The Golgi impregnation method: Unveiling morphological features of retinal cells Santiago Ramón y Cajal The retina of vertebrates (1892) The retina: a cornucopia of cell types Number of cell subpopulations RPE OS RPE cell: 1 IS OPL IPL Cone: Rod: Horizontal cell: Bipolar cell: Müller cell: Amacrine cell: > 26 GCL NFL Ganglion cell: 23 Astrocyte: 1 Microglial cell: 1 Primate retina 1

2 OUTLINE IHC on retina: technical aspects Cell-specific antibodies / features of disease Drug induced retinal remodeling: a case presentation OUTLINE IHC on retina: technical aspects Cell-specific antibodies / features of disease Drug induced retinal remodeling: a case presentation Why use IHC to examine the retina? Drug toxicity/disease may affect specific cell population To establish stages of disease (markers), kinetics of cell death Identify by-stander effect Retinal remodeling events (neurons, glial cells) Deciphering molecular mechanisms of disease/ drug toxicity Insights into functional alterations (e.g. protein mislocalization) Assessing response to therapy 2

3 Challenges of IHC in the retina Fixation protocol: which to choose? To trim or not to trim? Embedding media Sectioning Antigen retrieval? Immunoenzymatic vs Immunofluorescence detection? FIXATION PROTOCOLS - Aldehyde derivatives (by immersion and/or perfusion) paraformaldehyde (2%-4%) and/or glutaraldehyde (0.1%-2.5%) - Bouin s fixative - Davidson's fixative MORPHOLOGY ANTIGENICITY Bouin s Paraffin Paraformaldehyde OCT 1) Immersion of entire globe for 24 hrs 1) Slit 5 mm behind limbus 2) Fixation in 4% PF 3) Isolation of posterior cup (remove vitreous!) 2) Transfer to 70% EtOH (can be stored) 4) Trimming 5) Dehydration in gradient conc. of EtOH 4) Fixation in 4% PF/ 2% PF for 24 hrs 6) Trimming of posterior cup (if large eye) 6) Paraffin embedding 7) Dehydration in 15%-30% sucrose 8) Embedding in Optimal Cutting medium (OCT) 3

4 Sectioning Paraffin embedded tissue: 5-7 µm thickness OCT embedded tissue: µm thickness; (down to 3 µm with vacutome) Aspiration system (= Vacutome) Prevents rolling of cryosection Rolling of cryosection Antigen retrieval Prolonged aldehyde fixation protein cross-links Mask antigenic sites Heat induced epitope retrieval Retrieval solutions: Citrate buffer ph 6.0; Tris-EDTA ph 9.0; EDTA ph 8.0, Urea 5% Heat C for approx 20 min (Microwave, pressure cooker, Steamer) Proteolytic induced epitope retrieval Proteinase K, trypsin, chymotrypsin, pepsin, pronase, Determine optimal concentration and duration. No Ag retrieval With Ag retrieval (Courtesy Dr. C. Zeiss, Yale) Immunoenzymatic vs Immunofluorescence detection More possibilities of multiple labeling by immunofluorescence B Opsin R/G Opsin Rod opsin Merged RPE Autofluorescence in RPE of aged primates Pretreat with Na Borohydride (1% NaBH4) Switch to immunoenzymatic method Sensitivity: Immunoenzymatic (ABC method) > Immunofluorescence Occasional non-specific labeling of Outer Segments with ABC method OS Negative control (I ary Ab omitted) Negative control (I ary Ab omitted) 4

5 OUTLINE IHC on retina: technical aspects Cell-specific antibodies / features of disease Drug induced retinal remodeling: a case presentation Cell-specific antibodies: A tool box to explore retinal disease RPE RPE cell Rod Cone Horizontal cell Rod bipolar cell Cone bipolar cell Amacrine cell Müller cell Ganglion cell Retinal Pigment Epithelium (RPE) Function Cell markers RPE65, CRALBP (also labels Müller cells), bestrophin, (From: Strauss O., Physiol Rev, 2005) RPE From: Guziewicz K., IOVS, 2007 RPE65 CRALBP Bestrophin 5

6 IHC features of disease - Absence of labeling (RPE65 mutant) Wild type dog retina Retinal Pigment Epithelium (RPE) RPE65 -/- dog retina (From: Acland G. et al., Nat. Genet., 2005) - RPE cell or macrophage migration in subretinal space? RPE65 / CD18 Photoreceptors Function Phototransduction Cell markers Opsins (rod, B, R/G), cone arrestin, PDE, transducin rod opsin B opsin R/G opsin Retinal flatmount Cone arrestin/ B opsin Cone arrestin/ R/G opsin Photoreceptors IHC features of disease - Opsin mislocalization: an early marker of disease Focal patches of rod opsin mislocalization in a carrier of X-linked PRA B opsin mislocalization R/G opsin mislocalization (From: Beltran W. et al., IOVS., 2006) 6

7 IHC features of disease - rod neurite sprouting Photoreceptors OPL IPL Rod opsin/ synaptophysin Reported in several animal models of RD (Rho transgenic pig, Rdy cat, XLPRA2 dog), and human RP patients. (From: Beltran W. et al., IOVS., 2006) - cone neurite sprouting: Reported in the rd mouse (P8) and human patients with ARMD Horizontal cells Function: contribute to center-surround receptive field of 2 nd and 3 rd neurons cat Primate (From: Webvision) Cell markers parvalbumin, calbindin, calretinin, neurofilament 200kDa (cell processes) Parvalbumin Calbindin (Flatmount) Horizontal cells IHC features of disease - Flattening and loss of horizontal cell processes Calbindin Normal adult dog 41 week-old XLPRA2 dog (From: Beltran W. et al., IOVS., 2006) - Hypertrophy and loss of processes, but limited cell loss initially. NF-200 Calbindin wt mouse rd mouse (From: Strettoi E., PNAS, 2000) (From: Strettoi E., J. Neuroscience, 2003) 7

8 Function: Bipolar cells Second order neurons of the retina: Transfer of visual signals from PRs to GCs Morphology (Golgi stain): 1 type of rod BP, 9-10 different cone BP Cone BPs connect directly to GCs Rod BPs connect indirectly (via AII amacrine) to GCs Electrophysiology: ON BPs depolarize when light stimulates the center of their receptive field OFF BPs hyperpolarize when light stimulates the center of their receptive field Bipolar cells Cell markers PKCα (rod BP); Goα (ON BP), GRM6 (ON BP dendrites), NK3R (OFF-cone BP); recoverin (cone BP) PKCα Goα PKCα / Goα merged Bipolar cells IHC features of disease - Retraction of dendrites, reduction in cell number PKCα / Goα PKCα / Goα Early stage of disease Late stage of disease Focal retraction of rod bipolar cell dendrites in carrier of XLPRA Reported in: rd, XLPRA2, and human RP patients? - - Dendrite outgrowth into the (cat model of retinal detachment) 8

9 Amacrine cells Function: Horizontal integration in the IPL of visual information between bipolar cell terminals, ganglion cell dendrites and other amacrine cells. Vast collection of amacrine subtypes (> 26) + Different kind of synaptic partners (BPs, GCs) + > 30 Different neurotransmitters (GABA, Glycine, Dopamine, Ach, serotonine, taurine, VIP, ) Different functions Cell markers GABA, GLYT1, Disabled-1, ChAT, TH, parvalbumin, calretinin IPL a b GCL GABA Dab-1 (AII amacrine) Amacrine cells IHC features of disease - No evidence of decreased density in PR degeneration GABA - In glaucoma/ischemia : Debate: decreased density or decreased immunoreactivity? Ganglion cells Function Collect visual information processed in the retina and send it to the brain (dlgn, supraoptic and subthalamic structures) via NFL, and optic nerve. Cell markers Brn3a, NF-200, BDNF, TrkB M cells P cells GCL Brn3a (Cat: area centralis) Cat: retinal periphery) Brn3a (Flatmount) 9

10 Ganglion cells NFL Labeling of GC soma Labeling of GC axon bundles NF-200 (Courtesy Dr. E. Vecino, Univ. Basque Country, Spain ) Features of disease - Decreased density of GCs (Glaucoma) Fluoro-Gold retrograde labeling of GCs (From: Vecino E., Exp Eye Res, 2005) - - Neurite sprouting (In retinal detachment/reattachment) Functions Müller cells Cell markers CRALBP, Glutamine Synthetase (GS), cyclin D3, Vimentin, GFAP (From: Bringmann S., Prog Ret Eye Res, 2006) CRALBP Glutamine synthetase (Courtesy Dr. E. Vecino) Vimentin Müller cells IHC features of disease - GFAP, Vimentin increase; CRALBP, GS decrease GFAP labeling of astrocytes in normal control retina GFAP labeling of reactive Müller cells during photoreceptor degeneration - Extension into subretinal space (in detached retina) - Extension on vitreal surface of retina (in reattached retina) (From: Fisher S., Vision Res, 2003) 10

11 Astrocytes Glial cell population present in NFL. In contact with vessels and/or GCs axons GFAP GFAP (flatmount) Microglial cells Phagocytic cells usually found in small number in NFL. Under pathologic conditions can be seen anywhere in the retina Markers: CD11b (rodents) (Lectin histochemistry; Note: also label macrophages): - Isolectin B4 Griffonia simplicifolia (for cat, rabbit, squirrel retina) - Ricinus communis agglutinin I (for human retina) OUTLINE IHC on retina: technical aspects Cell-specific antibodies / features of disease Drug induced retinal remodeling: a case presentation Purpose of Study: Determine the efficacy of CNTF in rescuing photoreceptors in XLPRA2 XLPRA2 Frameshift mutation in RPGRORF15 Hypoth: Toxic gain of function? Early onset and rapidly progressive retinal degeneration CNTF = Ciliary Neurotrophic Factor Intravitreal injection Rescues photoreceptors in several animal models of retinitis pigmentosa (mouse, rat, cat, dog) 11

12 Age(weeks) Stages of photoreceptor degeneration in XLPRA2 OS IS OS IS Normal (5 weeks) Affected Stage 1 (5 weeks) Affected Stage 2 (8 weeks) Affected Stage 3 (16 weeks) Affected Stage 4 (26 weeks) Affected Stage 5 (40.6 weeks) Superior (# nuclei of (number) nuclei) SUPERIOR Age (in weeks) (From: Beltran W. et al., IOVS., 2006) Kinetics of cell death in XLPRA2 Ora Optic disc Ora TUNEL positive PRs Optic disc TUNEL assay: Labels cells undergoing cell death. # of TUNEL cells / 10 6 µm 2 of Risk of Photoreceptor cell death Age (weeks) (From: Beltran W. et al., IOVS., 2006) CNTF does not rescue photoreceptors in XLPRA2 PBS CNTF (Injection 7 & 10 Weeks End 14 Weeks) 12

13 CNTF causes peripheral retina remodeling (XLPRA2, 8 wk, Tx grp#1) PBS CNTF OS IS CNTF causes increase thickness, and OS / IS loss in the periphery CNTF causes remodeling of photoreceptor cells XLPRA2, 12 weeks (Injections at 4 & 8 weeks) PBS CNTF Rod opsin / Cone arrestin Increased number of rods; Loss of rod & cone IS/OS; Cone nuclear kinesis; Neuronal ectopia (rods) CNTF causes remodeling of bipolar cells cells XLPRA2, 12 weeks (Injections at 4 & 8 weeks) PBS CNTF PKCα / Goα Neuronal ectopia of rod bipolar cells and dendritic sprouting. 13

14 CNTF causes remodeling of horizontal cells XLPRA2, 12 weeks (Injections at 4 & 8 weeks) PBS CNTF Calretinin Dendritic sprouting. CNTF causes remodeling of Müller cells XLPRA2, 12 weeks (Injections at 4 & 8 weeks) PBS CNTF CRALBP Nuclear kinesis. CNTF downregulates phototransduction proteins XLPRA2, 5 weeks (1 week post injection) Rod opsin / Cone arrestin Transient decrease in rod opsin and cone arrestin expression Early stage of cone nuclear kinesis 14

15 CNTF causes cell proliferation BrdU Ki67 CONCLUSION Increasing number of cell specific antibodies commercially available IHC complements information provided by histological staining for assessment of retinal toxicity/disease. IHC can be done on eyes/retinas fixed for routine histology, however may require optimizing tissue fixation/embedding. Use of 2-Photon excitation microscopy enables (live) deep-tissue imaging 15