Changes in Kir4.1 expression following CCI of the ION. (A1-A3) 10 days following. CCI of the ION there is a significant decrease in the expression of Kir4.1 in the trigeminal ganglion ipsilateral to the nerve injury and an increase in expression of the cell injury marker ATF3.

Changes in Kir4.1 expression 5 days after Kir4.1 dsRNA or globin dsRNA was injected into the trigeminal ganglion. There is a significant decrease in the expression of Kir4.1 in the Kir41. dsRNA injected ganglia and no change in the globin dsRNA injected ganglia. I, ipsilateral ganglion, C, contralateral ganglion.

(A and B) Nissl stained sections from Kir4.1 dsRNA (A) and globin dsRNA (B)-treated ganglia. There is no difference in the number of neurons ipsilateral to the injection between Kir4.1 dsRNA (20±1 neurons/100,000 μm²) and globin dsRNA (19±1 neurons/100,000 μm²) groups (ANOVA F=2.1, P=0.180 [n=3]). (C to H) Neurons (arrows) are immunolabeled for substance-P (C and D, red fluorescence) or IB4 (E and F, green fluorescence) or CGRP (G and H blue fluorescence) in Kir4.1 dsRNA (C, E and G) and globin dsRNA (D, F and H)-treated ganglia. There is no difference in the percentage of substance-P or IB4 positive neurons ipsilateral to the injection between Kir4.1 dsRNA (substance-P: 12±1%; IB4: 32±1%; CGRP 34%±1%) and globin dsRNA (substance-P: 14±1%; IB4: 33±1%; CGRP 32%±1%) groups (ANOVA substance-P: F=2.3, P=0.204; IB4: F=0.3, P=0.815; CGRP F=1.1, P=0.566 [n=3]). Scale bars: (A-H) 60 μm.

(A) Kir4.1 dsRNA injection into the trigeminal ganglion results in microglia activation (OX-42 immunostaining) in the brainstem. The arrow and arrowhead indicated the regions shown at high magnification in (B) and (C) respectively. (D and E). There is no change in Substance P or NK1 receptor staining in the trigeminal nucleus caudalis following Kir4.1 suppression in the trigeminal ganglion. Sections of the brainstem from a rat with Kir4.1 dsRNA injected into the left trigeminal ganglion immunostained for substance-P (D) or NK1 (E). Densitometry quantification of the immunostaining (arrows) shows no difference in the ipsilateral/contralateral pixel ratio between Kir4.1 dsRNA (substance-P: 1.2±0.1; NK1: 1.0±0.1) and globin dsRNA (substance-P: 0.9±0.1; NK1: 1.0±0.0) groups (ANOVA substance-P: F=2.4, P=0.315; NK1: F=0.5, P=0.531). Scale bars: (A) 2mm, (B and C) 50μm, (D and E) 1 mm.

(A1) Rats show increased sensitivity to von Frey hair testing beginning 1 day after Kir4.1 dsRNA injection. This sensitivity reaches a maximum at day 5. (A2) When compared to CCI rats there is no difference between groups. Note: in all graphs the values are taken at day 10 following the CCI when nociceptive scores were at the maximum value. (B1) Kir4.1 dsRNA reduces the number of licks per episode of drinking. (B2) The reduction in drinking behavior is similar to the CCI group. (C1) Following Kir4.1 dsRNA injection the number of eye closures on the injected side increases to a maximum on day 5 then declines thereafter. (C2) There is an increase from baseline for both Kir4.1 dsRNA-injected and CCI rats, but in the CCI group, the eye closure rate is greater than in the Kir4.1 dsRNA group. (A1, B1 and C1) #P<0.05, ##P<0.01, ###P<0.001 compared to pre-injection of dsRNA (implant); **P<0.01, ***P<0.001 compared to Kir4.1 dsRNA; ^P<0.05, ^^P<0.01, ^^^P<0.001 compared to contralateral side (n=10per group for Kir4.1 and globin for all comparisons). (A2 and C2) ##P<0.01, ###P<0.001 compared to ipsilateral side; (A2, B2 and C2) *P<0.05, **P<0.01, (C2) ***P<0.001 compared to appropriate control; ^^P<0.01 compared to Kir4.1 dsRNA. (n=10 per group for Kir4.1 and globin groups and n= 4 per group for CCI and Sham groups)

(A1) Injection of Kir4.1 dsRNA into the trigeminal ganglion inhibits the expression of Kir4.1 as shown by reduced SGC immunolabeling (green). (A2 and A3) Pseudocolor representation of the squares depicted in A1 shows that Kir4.1 inhibition is greatest close (A2) to the injection site indicated by the red fluorescent dye, DiI (arrow in A1) when compared to a distant area (A3). (B and C) Serial sections from a Kir4.1 dsRNA injection site showing that after the disappearance of Kir4.1 immunoreactivity (green, B), SGCs are still present as indicated by GLAST labeling (green, C). The asterisk (*) shows the same blood vessel on each section (B, C). (D) Kir5.1 immunolabeling (green) is still present in SGCs after Kir4.1 dsRNA injection (arrow shows DiI injection site). (E) Immunostaining for SK3 (green) from the region of the trigeminal ganglion injected with Kir4.1 dsRNA indicated by the presence of DiI (red, arrow). There is no reduction of the SGC-specific SK3. (F) Kir2.3 immunostaining (green) from the region of the trigeminal ganglion injected with Kir4.1 dsRNA indicated by the presence of DiI (red, arrow). There is no reduction of the Kir2.3 immunostaining. (G) Section from a Kir4.1 dsRNA injection site (arrows indicate DiI showing we are in the injection site) on day 14 showing the return of Kir4.1 expression (green). (H) Kir4.1 dsRNA injection site showing only a single ATF3 immunopositive neuron (arrow). The inset is a higher magnification of the ATF3 immunolabeled neuron. The asterisk (*) indicates the location of the center of DiI injection. (I) Large numbers of ATF3 positive neurons (arrows) are present after CCI of the ION. Scale bars: (A and H) 300 μm, (B and C) 90 μm, (D and I) 30 μm, (E) 60 μm (G and I) 180 μm.

(A) Nissl stained section showing trigeminal ganglion neurons (N) and the nuclei of surrounding SGCs (arrows). (B) 0.1μm semi-thin Epon section showing closely packed neurons (N) with surrounding SGCs (arrows). (C) Electron micrograph demonstrating that the darkly stained, often attenuated SGCs cytoplasm (arrow, arrowheads) closely surrounds the primary sensory neurons (N1, N2). The separation between the neuronal and SGC membranes is approximately 20 nm. (D) SGCs are immunopositive for Kir4.1. The unstained nuclei of SGCs (arrows) give a characteristic signet-ring appearance. N indicates the cell bodies of sensory neurons. (E1 to E3) Double immunolabeling with Kir4.1 (green, E1) and the glial-specific glutamate transporter (GLAST, red, E2) show extensive overlap in the merged image (yellow, E3) confirming that Kir4.1 is confined to SGCs. The arrow indicates the same SGC nucleus in each image. (F) Immunolabels for Kir4.1 (green) and the neuronal marker NF160 (red) do not overlap. Arrows indicate unstained SGC nuclei. (G and H) SGCs in rat (G) and primate (H) dorsal root ganglion are also Kir4.1 immunopositive. (I) Section of control trigeminal ganglion immunostained for Kir4.1 (green, arrow) and blood vessels (RECA-1 antibody, red, arrowheads). There is no overlap of the two labels. (J) Control trigeminal ganglion immunostained for Kir4.1 (red) and nuclei (DAPI, blue). The only Kir4.1 immunopositive structures are the SGCs surrounding unstained neurons (N). Nuclei (arrows) in the surrounding unstained regions belong to Schwann cells and the outline of unstained myelin sheaths (arrowheads) is visible. Scale bars: (A, D, G, H, I and J) 30 μm, (B, E and F) 15 μm, (C) 5 μm.