Supplemental figures

(1)

Fig. S1. (A) Timeline and representative image of ipsilateral-LC (LC

ipsi

) and contralateral-LC

(LC

contra

) inhibition with a lidocaine (4%) microinjection in Sprague-Dawley rats. (B)

Representative image of LC

ipsi

inhibition by lidocaine (4%) microinjection. The graphs show the

withdrawal threshold of the ipsilateral and contralateral paw of ST-, MT- and LT-CCI rats in

pressure tests (ST - Sham-Sal n=4, Sham-Lido n=4, CCI-Sal n=4, CCI-Lido n=3; MT - Sham-

Sal n=4, Sham-Lido n=4, CCI-Sal n=4, CCI-Lido n=4; LT - Sham-Sal n=4, Sham-Lido n=4,

CCI-Sal n=3, CCI-Lido n=4: ***p<0.001 vs Sham-Sal; +p<0.05 vs CCI-Sal). (C)

Representative image of LC

contra

inhibition by lidocaine (4%) microinjection. The graphs show

response of the Ipsilateral and Contralateral paw of ST-, MT- and LT-CCI rats in the acetone

test (ST - Sham-Sal n=8, Sham-Lido n=10, CCI-Sal n=8, CCI-Lido n=10; MT - Sham-Sal n=9,

Sham-Lido n=10, CCI-Sal n=10, CCI-Lido n=10; LT - Sham-Sal n=7, Sham-Lido n=9, CCI-Sal

n=9, CCI-Lido n=10: ***p<0.001 vs Sham-Sal; +p<0.05, +++p<0.001 vs CCI-Sal). CCI,

Chronic constriction injury; PP, paw pressure; Sal, Saline; Lido, Lidocaine; Ipsi-paw, ipsilateral

paw; Contra-paw, contralateral paw.

(2)

Fig. S2. (A) Representative image of LC

contra

inhibition by lidocaine (4%) microinjection in Sprague-Dawley rats. Photomicrograph of a coronal section (neutral red stain) of a Sprague- Dawley rat brainstem showing the spread of Pontamine Sky Blue solution after its administration into the LC (scale bar: 250 µm) and a higher magnification of the inset (scale bar 250 µm). (B) Schemes adapted from the Paxinos and Watson atlas (co-ordinates relative to bregma: Paxinos and Watson, 2007) to show the lidocaine microinjection sites within (in) and beyond (out) the LC of the LT-CCI rats. The graph shows the duration of the effect of lidocaine in the acetone test, the graph shows the response of the ipsilateral hindpaw 15 and 120 min after applying acetone (CCI-Sal n=9, CCI-CNO n=10: ***p<0.001 vs CCI-Lido (15’); +++p<0.001 vs CCI-Sal (15’). The next graph shows the response of the ipsilateral paw in the acetone test when lidocaine was microinjected outside the LC (CCI-Sal [out-LC] n=3, CCI-Sal [in-LC] n=9, CCI-CNO [in-LC] n=10: +++p<0.001 vs CCI-Sal). CCI, Chronic constriction injury; Lido, lidocaine; Sal, saline; LC, locus coeruleus; 4V, fourth ventricle; Ipsi-paw, ipsilateral paw;

Contra-paw, contralateral paw.

(3)

Fig. S3. (A) Timeline of contralateral-LC (LC

contra

) inhibition with a lidocaine (4%) microinjection in Long-Evans rats. (B) Representative image of LC

contra

inhibition by lidocaine (4%) microinjection. The graphs show the withdrawal threshold of the ipsilateral and contralateral paw of CCI-LT rats in acetone and paw pressure test (CCI-Sal n=6, CCI-Lido n=8). (C) Quantification of pCREB positive neurons to the ipsilateral DRt (DRt

ipsi

) and contralateral (DRt

contra

) of sham and CCI-LT rats of Long-Evans rats (Sham n=4, CCI n=4). (D) Representative image of pCREB positive neurons into the DRt

ipsi

or DRt

contra

of CCI-LT rats.

CCI, Chronic constriction injury; LT, Long-term; LC, locus coeruleus; DRt, dorsal reticular

nucleus; IR, immunoreactivity; A, acetone; PP, paw pressure; Sal, Saline; Lido, Lidocaine; Ipsi-

paw, ipsilateral paw; Contra-paw, contralateral paw. Some elements of this figure were

produced using BioRender (www.BioRender.com).

(4)

Fig. S4. Representative immunofluorescence of DBH, mCherry and GFP expression in (A-D)

locus coeruleus neurons (E) A5 noradrenergic nucleus and (F) parabrachial nucleus (no

noradrenergic). Scale bar: 20 μm.

(5)

Fig. S5. (A) Timeline and scheme of the retrograde CAV-Cre-GFP injection into the DRt

contra

and AAV-mCherry (control virus) into LC

contra

of Sprague-Dawley rats to test that CNO does

not provoke changes in the nociceptive tests. (B) The graphs show the response of the Ipsilateral

and Contralateral paw in the acetone, von Frey and spontaneous pain tests after CNO (1 mg/kg,

i.p.) administration to CCI-LT animals (CCI-Sal n=8; CCI-CNO n=5). (C) The graphs show the

response of the Ipsilateral and Contralateral paw in the acetone, von Frey and spontaneous pain

(6)

tests after clonidine (20 µg, i.t.) administration (CCI-Sal n=8; CCI-Cloni n=5): +++p<0.001 vs CCI-Sal. (D) Representative image of the intrathecal injection of Pontamine Sky Blue solution.

CCI, Chronic constriction injury; LC, locus coeruleus; DRt, dorsal reticular nucleus; LT, Long- term; CNO, clozapine-n-oxide; Sal, Saline; Cloni, clonidine; A, acetone test; VF, von Frey test;

SP, spontaneous pain test; i.t., intrathecal; i.p., intraperitoneal; Ipsi-paw, ipsilateral paw; Contra-

paw, contralateral paw.

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Table 1. Statistical analysis for Fig.1A

➢ Shapiro-Wilk normality test:

Ipsilateral paw ST (CCI-2d) MT (CCI-7d) LT (CCI-28d)

Acetone test Sham CCI Sham CCI Sham CCI

W 0,921 0,851 0,845 0,776 0,825 0,891

P value 0,396 0,077 0,065 0,011 0,039 0,204

Passed normality test

(alpha= 0.05)? Yes Yes Yes No No Yes

Contralateral paw ST (CCI-2d) MT (CCI-7d) LT (CCI-28d)

Acetone test Sham CCI Sham CCI Sham CCI

W 0,655 0,638 0,909 0,963 0,917 0,795

P value 0,000 0,000 0,308 0,830 0,364 0,018

(alpha= 0.05)? No No Yes Yes Yes No

➢ Non-parametric test:

ST (CCI-2d) MT (CCI-7d) LT (CCI-28d)

Acetone Test Ipsilateral paw Contralateral paw Ipsilateral paw Contralateral paw Ipsilateral paw Contralateral paw Mann-Whitney test:

P value 0,001** 0,090 0,000*** 0,833 0,000*** 0,525

Mann-Whitney U 6 20,5 0 38 0 32,5

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Table 2. Statistical analysis for Fig.1C and Fig. S1B

➢ Shapiro-Wilk normality test

Acetone test Sham-Sal Sham-Lido CCI-Sal CCI-Lido Sham-Sal Sham-Lido CCI-Sal CCI-Lido Sham-Sal Sham-Lido CCI-Sal CCI-Lido

W 0,971 0,727 0,901 0,824 0,640 0,853 0,884 0,870 0,601 0,724 0,864 0,951

P value 0,906 0,003 0,257 0,051 0,001 0,167 0,208 0,187 0,000 0,004 0,107 0,697

(alpha= 0.05)? Yes No Yes Yes No Yes Yes Yes No No Yes Yes

Paw Pressure test

W 0,863 0,920 0,971 1 0,895 0,895 0,993 0,981 0,827 0,920 0,855 0,887

P value 0,272 0,539 0,85 >0,999 0,406 0,406 0,972 0,911 0,161 0,538 0,253 0,369

(alpha= 0.05)? Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

W 0,889 0,916 0,684 0,827 0,907 0,915 0,801 0,858 0,782 0,693 0,617 0,813

P value 0,194 0,364 0,001 0,055 0,415 0,473 0,030 0,144 0,018 0,002 0,000 0,028

(alpha= 0.05)? Yes Yes No Yes Yes Yes No Yes No No No No

W 0,863 0,963 0,971 0,964 0,827 0,998 0,971 0,916 0,993 0,827 1 0,939

P value 0,272 0,798 0,85 0,637 0,161 0,995 0,85 0,513 0,972 0,161 >0,999 0,650

(9)

➢ Non-parametric test

Acetone Test Ipsilateral paw Contralateral paw Ipsilateral paw Contralateral paw Ipsilateral paw Contralateral paw Kruskal-Wallis test:

P value <0,000*** 0,286 0,000*** 0,569 <0,000*** 0,566

Kruskal-Wallis statistic 27,27 3,785 19,86 2,015 25,36 2,029

Mann-Whitney test:

Sham-Sal vs Sham-Lido

P value 0,075 0,196 0,688 0,794 0,928 0,922

Mann-Whitney U 20 25 14 15 28,5 29

Sham-Sal vs CCI-Sal

P value 0,002** 0,195 0,000*** 0,667 <0,000*** 0,413

Mann-Whitney U 6,5 25 0 20 0 27

CCI-Sal vs CCI-Lido

P value 0,001** 0,767 0,536 >0,999 0,992 0,250

Mann-Whitney U 5,5 33 22,5 28 40 25,5

➢ Parametric test

Two-way ANOVA: ST (CCI-2d) MT (CCI-7d) LT (CCI-30d)

Paw Pressure test Ipsilateral paw Contralateral paw Ipsilateral paw Contralateral paw Ipsilateral paw Contralateral paw Surgery F(1,11)=164,159*** F(1,11)=0,173 F(1,12)=107,391*** F(1,12)=0,000 F(1,11)=60,172*** F(1,11)=0,688 Treatment F(1,11)=10,743** F(1,11)=0,530 F(1,12)=0,023 F(1,12)=6,000 F(1,11)=0,164 F(1,11)=0,416 Interaction F(1,11)=4,350 F(1,11)=3,908 F(1,12)=6,519* F(1,12)=0,050 F(1,11)=0,335 F(1,11)=1,910

(10)

Table 3. Statistical analysis for Fig.1D and Fig. S1C

W 0,736 0,825 0,919 0,939 0,889 0,892 0,838 0,907 0,818 0,947 0,939 0,924

P value 0,006 0,029 0,424 0,541 0,194 0,177 0,042 0,259 0,062 0,663 0,571 0,391

(alpha= 0.05)? No No Yes Yes Yes Yes No Yes Yes Yes Yes Yes

W 0,856 0,823 0,913 0,968 0,965 0,974 0,895 0,819 0,911 0,945 0,867 0,907

P value 0,11 0,028 0,374 0,873 0,848 0,926 0,194 0,025 0,400 0,637 0,113 0,260

(alpha= 0.05)? Yes No Yes Yes Yes Yes Yes No Yes Yes Yes Yes

W 0,798 0,872 0,877 0,858 0,874 0,879 0,911 0,904 0,820 0,841 0,883 0,769

P value 0,027 0,105 0,178 0,073 0,137 0,127 0,287 0,245 0,064 0,059 0,170 0,006

(alpha= 0.05)? No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No

W 0,968 0,919 0,983 0,929 0,866 0,896 0,961 0,941 0,868 0,920 0,977 0,884

P value 0,882 0,352 0,975 0,437 0,111 0,197 0,793 0,560 0,179 0,396 0,946 0,146

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Acetone test Ipsilateral paw Contralateral paw Ipsilateral paw Contralateral paw Ipsilateral paw Contralateral paw Kruskal-Wallis test:

P value 0,002** 0,183 <0,000*** 0,307

Kruskal-Wallis statistic 14,850 4,847 24,530 3,604

Mann-Whitney test:

P value >0,999 0,163 0,609 0,292

Mann-Whitney U 38,5 25 38 21

P value 0,000*** 0,076 <0,000*** 0,472

Mann-Whitney U 1,5 15,5 0,5 24,5

P value 0,344 0,997 0,002** 0,249

Mann-Whitney U 29 39 10,5 31

Paw pressure test Kruskal-Wallis test:

P value 0,005** <0,000***

Kruskal-Wallis statistic 12,87 30,67

Mann-Whitney test:

P value 0,113 0,082

Mann-Whitney U 22 23,5

P value 0,044* <0,000***

Mann-Whitney U 13 0

P value 0,840 0,031*

Mann-Whitney U 37,5 22

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Two-way ANOVA ST (CCI-2d) MT (CCI-7d) LT (CCI-30d)

Acetone test Ipsilateral paw Contralateral paw Ipsilateral paw Contralateral paw Ipsilateral paw Contralateral paw

Surgery F(1,35)=1,537 F(1,31)=22,141***

Treatment F(1,35)=0,622 F(1,31)=21,455***

Interaction F(1,35)=0,001 F(1,31)=21,962***

Paw pressure test

Surgery F(1,32)=3,564 F(1,35)=2,446 F(1,31)=173,447*** F(1,31)=2,611

Treatment F(1,32)=2,917 F(1,35)=0,612 F(1,31)=35,392*** F(1,31)=0,012

Interaction F(1,32)=0,655 F(1,35)=1,184 F(1,31)=21,401*** F(1,31)=1,518

Table 4. Statistical analysis for Fig. S2

CCI-sal (15') CCI-Lido (15') CCI-sal (120') CCI-Lido (120')

Acetone test

W 0,939 0,924 0,968 0,900

P value 0,571 0,392 0,875 0,216

Passed normality test (alpha= 0.05)? Yes Yes Yes Yes

Parametric test

Acetone test

One-way ANOVA with repeated measure:

Treatment F(1,17)=12,335**

Time F(1,17)=5,893*

Treatment x Time F(1,17)=13,637**

Unpaired Student’s t-test:

CCI-Lido vs CCI-Sal (in-LC) t (17)=5,710***

CCI-Lido vs CCI-Sal (out-LC) t (10)=0.609

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Table 5. Statistical analysis of Fig. S3B

Ipsilateral paw Contralateral paw

Acetone test CCI-Sal CCI-Lido CCI-Sal CCI-Lido

W 0,908 0,843 0,701 0,418

P value 0,421 0,082 0,006 <0,0001

Passed normality test (alpha= 0.05)? Yes Yes No No

W 0,916 0,920 0,876 0,934

P value 0,473 0,433 0,252 0,551

Acetone test Ipsilateral paw Contralateral paw

Mann-Whitney test:

P value 0,539

Mann-Whitney U 20

➢ Parametric tests

Acetone test

Unpaired Student’s t-test: t(12)=0,874

Paw pressure test

Unpaired Student’s t-test: t(12)=1,139 t(12)=0,143

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Table 6. Statistical analysis for Fig. 2A

Immobility Climbing Swimming

Forced swimming test Sham CCI Sham CCI Sham CCI

W 0,791 0,866 0,993 0,891 0,801 0,907

P value 0,087 0,210 0,972 0,324 0,103 0,415

(alpha= 0.05)? Yes Yes Yes Yes Yes Yes

Forced swimming test Immobility Climbing Swimming Unpaired Student’s t-

test: t(8)=11*** t(8)=14,06*** t(8)=0,388

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Table 7. Statistical analysis for Fig. 2B

➢

Shapiro-Wilk normality test

Ipsilateral LC Immobility Climbing Swimming

Forced swimming test Sham-Sal Sham-Lido CCI-Sal CCI-Lido Sham-Sal Sham-Lido CCI-Sal CCI-Lido Sham-Sal Sham-Lido CCI-Sal CCI-Lido

W 0,867 0,952 0,979 0,890 0,857 0,928 0,739 0,917 0,822 0,850 0,914 0,914

P value 0,214 0,760 0,928 0,272 0,178 0,566 0,023 0,448 0,091 0,158 0,490 0,421

(alpha= 0.05)? Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes

Contralateral LC Immobility Climbing Swimming

Forced swimming test Sham-Sal Sham-Lido CCI-Sal CCI-Lido Sham-Sal Sham-Lido CCI-Sal CCI-Lido Sham-Sal Sham-Lido CCI-Sal CCI-Lido

W 0,933 0,950 0,895 0,963 0,905 0,938 0,843 0,922 0,896 0,591 0,843 0,887

P value 0,479 0,671 0,191 0,820 0,247 0,531 0,048 0,371 0,198 <0,0001 0,048 0,157

(alpha= 0.05)? Yes Yes Yes Yes Yes Yes No Yes Yes No No Yes

Ipsilateral LC Contralateral LC

Forced swimming test Immobility Climbing Swimming Immobility Climbing Swimming

Kruskal-Wallis test:

P value 0,007** 0,001** 0,715

Kruskal-Wallis statistic 12,03 16 1,359

Mann-Whitney test:

P value 0,429 0,516 0,676

Mann-Whitney U 12,5 41 44

P value 0,004** 0,008** 0,490

Mann-Whitney U 0 16 40,5

P value 0,003** <0,000*** 0,667

Mann-Whitney U 0 0 44

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Forced swimming test Ipsilateral LC Contralateral LC

Two-way ANOVA: Immobility Climbing Swimming Immobility Climbing Swimming

Surgery F(1,20)=8,735** F(1,20)=0,073 F(1,36)=6,191*

Treatment F(1,20)=3,627 F(1,20)=0,025 F(1,36)=7,510**

Surgery x Treatment F(1,20)=11,865** F(1,20)=2,540 F(1,36)=10,300**

Table 8. Statistical analysis for Fig. 2C

Contralateral LC Sham-Sal Sham-Lido CCI-Sal CCI-Lido Spontanous locomotor activity

W 0,969 0,903 0,912 0,922

P value 0,889 0,309 0,369 0,444

Passed normality test (alpha=

0.05)? Yes Yes Yes Yes

Spontanous locomotor activity Two-way ANOVA (repeated measure):

Surgery F(1,13)=0,330

Treatment F(1,13)=0,002

Surgery x Treatment F(1,13)=0,033

Time F(7,91)=83,612***

Surgery x Time F(7,91)=0,647

Treatment x Time F(7,91)=0,501

Surgery x Treatment x Time F(7,91)=0,536

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Table 9. Statistical analysis for Fig. 3B

➢ Shapiro-Wilk normality test Ipsilateral paw

Distribution FG (%) Ipsilateral LC Contralateral LC

W 0,947 0,947

P value 0,554 0,554

(alpha= 0.05)? Yes Yes

Distribution FG (%)

Unpaired Student’s t-test: t(4)=6,179**

Table 10. Statistical analysis for Fig. 3C

Ipsilateral LC Contralateral LC Distribution FG (%) Dorsal Ventral Dorsal Ventral

W 0,835 0,835 0,852 0,852

P value 0,202 0,202 0,245 0,245

(alpha= 0.05)? Yes Yes Yes Yes

Ipsilateral LC Contralateral LC

Distribution FG (%)

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Table 11. Statistical analysis for Fig. 3D

Shapiro-Wilk normality test

Ipsilateral paw contralateral paw

pCREB expression (DRt) Sham CCI Sham CCI

W 0,989 0,999 0,775 0,937

P value 0,953 0,953 0,064 0,516

Passed normality test (alpha=

0.05)? Yes Yes Yes Yes

Parametric test

Two-way ANOVA:

pCREB expression (DRt)

Surgery F(1,10)=4,275

Side (LC) F(1,10)=5,581*

Surgery x Side (LC) F(1,10)=15,994**

Table 12. Statistical analysis of Fig. S3C

Ipsilateral paw contralateral paw pCREB expression into DRt Sham CCI Sham CCI

W 0,989 0,999 0,775 0,937

P value 0,953 0,953 0,064 0,516

Two-way ANOVA:

pCREB expression into DRt

Surgery F(1,12)=0,588

LC F(1,12)=0,036

Surgery x LC F(1,12)=5,799

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Table 13. Statistical analysis for Fig. 4B

Acetone test Sham-Sal Sham-CNO CCI-Sal CCI-CNO Sham-Sal Sham-CNO CCI-Sal CCI-CNO

W 0,866 0,833 0,849 0,940 0,496 0,645 0,418 0,509

P value 0,212 0,086 0,093 0,549 <0,000 0,001 <0,000 <0,000

(alpha= 0.05)? Yes Yes Yes Yes No No No No

Von Frey test

W 0,943 0,938 0,938 0,908 0,940 0,829 0,954 0,852

P value 0,685 0,617 0,594 0,271 0,656 0,079 0,746 0,062

(alpha= 0.05)? Yes Yes Yes Yes Yes Yes Yes Yes

Spontaneous pain test

W 0,640 0,664 0,971 0,991 0,496 0,646 0,724 0,641

P value 0,001 0,002 0,908 0,998 <0,000 0,001 0,004 0,000

(alpha= 0.05)? No No Yes Yes No No No No

(20)

Acetone test Kruskal-Wallis test:

P value 0,270

Kruskal-Wallis statistic 3,918

Mann-Whitney test:

Sham-Sal vs Sham-CNO

P value <0.999

Mann-Whitney U 19,000

P value 0,528

CCI-Sal vs CCI-CNO

P value 0,261

Spontaneous pain test Kruskal-Wallis test:

P value <0,000*** 0,801

Mann-Whitney test:

P value >0,999 0,853

P value 0,001*** 0,497

P value 0,000*** >0,999

(21)

Two-way ANOVA: Ipsilateral paw Contralateral paw Acetone test

Surgery F(1,27)=168,304***

Treatment F(1,27)=41,967***

Surgery x Treatment F(1,27)=38,424***

Von Frey test

Surgery F(1,27)=91,314*** F(1,27)=0,486

Treatment F(1,27)=9,220** F(1,27)=0,701

Surgery x Treatment F(1,27)=7,249* F(1,27)=1,182

Table 14. Statistical analysis for Fig. 4C

Acetone test Sham-Sal Sham-Cloni CCI-Sal CCI-Cloni Sham-Sal Sham-Cloni CCI-Sal CCI-Cloni

W 0,552 0,496 0,932 0,820 0,552 0,640 0,566 0,650

P value 0,000 <0,000 0,534 0,025 0,000 0,001 <0,000 0,000

Passed normality test (alpha= 0.05)? No No Yes No No No No No

Von Frey test

W 0,983 0,931 0,964 0,909 0,893 0,990 0,899 0,882

P value 0,949 0,587 0,851 0,278 0,375 0,987 0,285 0,136

Passed normality test (alpha= 0.05)? Yes Yes Yes Yes Yes Yes Yes Yes

W 0,684 0,775 0,971 0,887 0,684 0,640 0,827 0,684

P value 0,006 0,035 0,908 0,184 0,006 0,001 0,055 0,001

Passed normality test (alpha= 0.05)? No No Yes Yes No No Yes No

(22)

Acetone test

P value <0,000*** 0,950

Mann-Whitney test:

Sham-Sal vs Sham-Cloni

P value >0.999 >0,999

P value 0,000*** >0,999

Mann-Whitney U 0 19

P value 0,000*** 0,843

Mann-Whitney U 0 37

P value <0,000**** 0,586

Mann-Whitney test:

Sham-Sal vs Sham-Cloni

P value 0,664 0,567

P value 0,000*** >0,999

Mann-Whitney U 0 18

CCI-Sal vs CCI-Cloni

P value 0,000*** 0,426

Mann-Whitney U 2,5 26,5

(23)

Two-way ANOVA: Ipsilateral paw Contralateral paw Von Frey test

Surgery F(1,25)=50,891*** F(1,25)=0,073 Treatment F(1,25)=6,858* F(1,25)=1,142 Interaction F(1,25)=3,899 F(1,25)=0,118

Table 15. Statistical analysis for Fig. S5B

Ipsilateral paw Contralateral paw Acetone test CCI-Sal CCI-CNO CCI-Sal CCI-CNO

W 0,849 0,953 0,418 0,552

P value 0,093 0,758 <0,000 0,000

(alpha= 0.05)? Yes Yes No No

Von Frey test

W 0,938 0,850 0,953 0,855

P value 0,594 0,194 0,746 0,211

W 0,971 0,849 0,724 0,771

P value 0,908 0,192 0,004 0,046

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Acetone test

Mann-Whitney test:

P value >0.999

Mann-Whitney U 18,5

Mann-Whitney test:

P value >0,999

Mann-Whitney U 19

Acetone test

Unpaired Student’s t-test: t(11)=0,531

Von Frey test

Spontaneous pain test Unpaired Student’s t-test: t(11)=0,989

(25)

Table 16. Statistical analysis for Fig. S5C

Acetone test CCI-Sal CCI-CNO CCI-Sal CCI-CNO

W 0,932 0,833 0,566 0,552

P value 0,534 0,146 <0,000 0,000

Von Frey test

W 0,964 0,864 0,899 0,914

P value 0,851 0,245 0,285 0,493

W 0,971 0,902 0,827 0,684

P value 0,908 0,421 0,055 0,006

(alpha= 0.05)? Yes Yes Yes No

Acetone test

Mann-Whitney test:

P value >0,999

Mann-Whitney U 19

Mann-Whitney test:

P value 0,499

Mann-Whitney U 14,5

(26)

Acetone test

Unpaired Student’s t-test: t(11)=11,64***

Von Frey test

Unpaired Student’s t-test: t(11)=6,311*** t(11)=1,291

Unpaired Student’s t-test: t(11)=5,575***

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Table 17. Statistical analysis for Fig. 5B

Immobility Climbing Swimming

Forced swimming test Sham-Sal Sham-CNO CCI-Sal CCI-CNO Sham-Sal Sham-CNO CCI-Sal CCI-CNO Sham-Sal Sham-CNO CCI-Sal CCI-CNO

W 0,809 0,960 0,897 0,869 0,809 0,960 0,905 0,864 0,366 0,390 0,486 0,392

P value 0,019 0,797 0,172 0,119 0,019 0,797 0,214 0,106 <0.000 <0.000 <0.000 <0.000

(alpha= 0.05)? No Yes Yes Yes No Yes Yes Yes No No No No

Ipsilateral LC

Forced swimming test Immobility Climbing Swimming Kruskal-Wallis test:

P value 0,023* 0,019* 0,816

Kruskal-Wallis statistic 9,494 9,993 0,937

Mann-Whitney test:

P value 0,007** 0,007** >0.999

P value 0,002** 0,000*** 0,563

Mann-Whitney U 13 10,5 49,50

P value 0,751 0,751 0,864

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Table 18. Statistical analysis for Fig. 5C

Sham-Sal Sham-CNO CCI-Sal CCI-CNO

Spontanous motor activity

W 0,916 0,910 0,906 0,880

P value 0,399 0,352 0,326 0,189

Spontanous motor activity

Two-way ANOVA (repeated measure):

Surgery F(1,35)=0,163

Treatment F(1,35)=0,515

Surgery x Treatment F(1,35)=0,192

Time F(7,245)=5,098***

Surgery x Time F(7,245)=0,983

Treatment x Time F(7,245)=0,769 Surgery x Treatment x Time F(7.245)=0.568

(29)

Table 19. Statistical analysis for Fig. 5D

20 min after CNO Ipsilateral paw Contralateral paw

W 0,859 0,684 0,952 0,952 0,731 0,684 0,655 0,794

P value 0,074 0,001 0,709 0,712 0,002 0,001 0,000 0,017

(alpha= 0.05)? Yes No Yes Yes No No No No

Von Frey test

W 0,925 0,914 0,852 0,944 0,840 0,870 0,934 0,899

P value 0,404 0,346 0,078 0,621 0,044 0,122 0,517 0,247

(alpha= 0.05)? Yes Yes Yes Yes No Yes Yes Yes

360 min after CNO Ipsilateral paw Contralateral paw

W 0,594 0,780 0,910 0,883 0,778 0,825 0,617 0,655

P value <0.000 0,012 0,314 0,169 0,008 0,039 0,000 0,000

(alpha= 0.05)? No No Yes Yes No No No No

Von Frey test

W 0,984 0,963 0,885 0,784 0,928 0,891 0,945 0,814

P value 0,984 0,826 0,178 0,014 0,428 0,205 0,633 0,030

(alpha= 0.05)? Yes Yes Yes No Yes Yes Yes No

(30)

20 min after CNO 360 min after CNO

Acetone test Ipsilateral paw Contralateral paw Ipsilateral paw Contralateral paw

P value <0.000*** 0,387 P<0,000*** 0,274

Kruskal-Wallis statistic 57,58 7.41 28,37 3,888

Mann-Whitney test:

P value 0.256 0950 0,550 0,661

Mann-Whitney U 31 42.50 36,5 40

P value <0.000*** 0,565 0,000*** 0,106

Mann-Whitney U 0 39 0 25,5

P value 0.001** 0,362 0,413 >0.999

Mann-Whitney U 6 29,5 31 36

Von Frey test Kruskal-Wallis test:

P value 0,850 <0,000*** 0,560

Kruskal-Wallis statistic 3,361 26,74 2,062

Mann-Whitney test:

P value 0,940 0,734 0,870

Mann-Whitney U 44,5 40,5 42,5

P value 0,840 0,000*** 0.988

P value 0,781 0,462 0,285

Mann-Whitney U 37 38,5 28

(31)

Two-way ANOVA:

Von Frey test 20 min Ipsilateral paw Contralateral paw

Surgery F(1,33)=225,011***

Treatment F(1,33)=4,750*

Interaction F(1,33)=6.528*