D .M. White Brain Research 885 2000 79 –86
81
of 30 sucrose in PBS. The lumbar spinal cord was partially ligated and osmotic pumps were attached to the
removed and stored overnight in 30 sucrose. Fifty catheters and implanted subcutaneously. The results are
micron, transverse sections were cut on a freezing mi- expressed as the percentage change in mechanical thres-
crotome. Sections were reacted for HRP using tetra- hold of the injured paw as compared to the sham-operated
methylbenzidine as the chromagen, dehydrated, cleared control and calculated as: hthreshold of injured paw2
and mounted. The density of terminal labelling across the threshold of sham-operated paw3100j threshold of
different laminae was determined by image analysis. Five sham-operated paw.
groups of animals were examined. These were: 1 un- treated controls; 2 sham operated; 3 nerve transection;
2.5. Statistics 4 nerve-transected animals treated intrathecally with NT-
3 antisense oligonucleotide; and 5 nerve-transected ani- Statistical analysis of the data was done using either
mals treated intrathecally with NT-3 sense. The density of one-way analysis of variance ANOVA followed by
labelling was determined by image analysis using a ´
Scheffe test post hoc comparisons, or Student’s t-test. previously published method [1]. The image was captured
directly from the microscope using a JVC digital camera. Four boxes 25325 mm were placed over the image
3. Results
within lamina II and in the adjacent lamina III area. The area of C-HRP label within these boxes was computed
3.1. Effect of NT-3 antisense oligonucleotide on nerve using Scion Image Windows version of NIH Image; Scion
injury-induced A b-fibre sprouting
Corp., MA. An average of the four measurements was calculated and the results are expressed as the ratio of
In sham-operated controls n55, C-HRP label is pres- lamina II lamina III. Transganglionic labelling of Ab-
ent in laminae I, III and deeper lamina, but not present in fibres was also done on two groups of animals with partial
lamina II Fig. 1A. This distribution of C-HRP is con- nerve ligation. In one group the C-HRP was injected
sistent with previous transganglionic labelling studies proximal to the ligature to label all Ab-fibres and, in the
using normal animals [1,35]. The image analysis data also second group, the conjugated was injected distal to the
shows that the density of C-HRP label in lamina II of ligature to label the intact fibres.
sham-operated animals is not different compared to un- In control experiments, the cross-sectional area of cells
treated animals Fig. 2; n54. Two weeks following labelled with C-HRP was examined in L5 dorsal root
complete transection of the sciatic nerve n55, C-HRP ganglion cells ipsilateral and contralateral to complete
label expands into lamina II Fig. 1B and the density is transection of the sciatic nerve n54. Twenty-micron
significantly different from both untreated and sham rats serial sections were cut, mounted and processed as de-
Fig. 2; ANOVA, P,0.05. scribed above. Cross-sectional area of individually labelled
Intrathecal administration of NT-3 antisense oligonu- cells was determined in every 20th section. The circumfer-
cleotide for 14 days via an osmotic minipump significantly ence of labelled cells, in which the nucleus could be seen,
attenuated the density of C-HRP label in lamina II of was manually drawn using a computer mouse and from
injured rats n54 compared to the control group of this the cross-sectional area was computed.
animals with complete nerve transection Figs. 1C and 2; nerve injury vs. nerve injury1antisense, P,0.05. Control
2.4. Behavioural test experiments show that intrathecal administration of sense
oligonucleotide n54 had no effect on the nerve injury- The nociceptive flexion reflex was quantified using an
induced expansion of C-HRP into lamina II Figs. 1D and Ugo Basile Analgesymeter Comerio-Varese, Italy. A
2. mechanical force increasing linearly with time was applied
A comparison of the cross-sectional area of cells la- to the dorsum of the rat’s hindpaw by a dome-shaped
belled with C-HRP reveals there is no significant differ- plunger diameter 1.4 mm, radius of curvature 368. The
ence in size of injured DRG cells compared to control cells nociceptive threshold was defined as the force, in grams, at
2
of the contralateral DRG injured, 1062619 mm , 528 which the rat withdrew its paw. The chronic intrathecal
2
profiles, n54; control, 1086624 mm , 404 profiles, n54. catheters were inserted 1 week before commencing be-
havioural studies to allow rats sufficient time for recovery. Nociceptive thresholds were then determined on a daily
3.2. Effect of NT-3 antisense oligonucleotide on nerve basis over a 3-week period. Each day, thresholds were
injury-induced allodynia determined at 10-min intervals for a period of 2 h. The
mean of the last six measurements represented the nocicep- The influence of NT-3 antisense and sense oligonucleo-
tive threshold for that day. After the first 5 days of testing, tides on nerve injury-induced allodynia was also examined
the rats were re-anaesthetized, the sciatic nerve was in animals with partial nerve ligation. Partial nerve ligation
82 D
Fig. 1. Photomicrographs of dorsal horn of lumbar spinal cord showing area of termination of Ab-fibres transganglionically labelled with C-HRP injected into the left sciatic nerve. A In sham-operated animals n55, C-HRP label is present in lamina I, III and deeper laminae. No C-HRP label is present in
lamina II . B Two weeks following complete sciatic nerve transection n55, C-HRP label expands into lamina II. C Intrathecal administration of NT-3 antisense oligonucleotides via osmotic pumps for 14 days n54 attenuated the intensity of C-HRP label in lamina II in nerve injured animals. D
Intrathecal administration of NT-3 sense n54 had no influence on intensity of C-HRP label in lamina II of rats with nerve injury. Calibration bar550 mm.
induces a significant decrease in threshold to mechanical C-HRP distal to the ligature also results in expansion of
stimulation compared to measurements determined prior to label in lamina II, suggesting sprouting occurs in intact
ligation in week 1 Fig. 3; P,0.0002, n58. Animals Ab-fibres Fig. 4C; n53.
treated intrathecally with NT-3 antisense significantly attenuated the nerve injury-induced allodynia compared to
the untreated control group of animals Fig. 3A; P,0.05, n510. The analgesic effect of the NT-3 antisense became
4. Discussion
