Indian Journal of Experimental Biology Vol. 40, August 2002, pp. 882-888

Role of adenosine in drug-induced catatonia in mice

Amanpreet Singh & S K Kulkarni*

Pharmacology Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, 160014, India

Received 11 June 2001 ; revised 21 August 2001

Parkinson's disease is one of the most common neurodegenerative disorders affecting large majority of population who are older than age of 65 . Apart from dopamine, acetylcholine and glutamate, adenosine has also been identified in the basal ganglia. Adenosine modulates the release of a variety of neurotransmitters including dopamine. In order to establish adeno­sine-dopamine interactions in drug-induced catatonia we studied the effect of adenosine in drug-induced catatonia in mice. [n the present study adenosine dose dependently produced catatonia when assessed on rota-rod and bar tests in mice. Adeno­sine also potentiated the catatonic effect of perphenazine. L-dopa plus carbidopa or OR-486 (a potent centrally acting COMT inhibitor) completely reversed adenosine-induced catatonia. Since reversal by scopolamine of adenosine-induced catatonia was not to the same extent as with I-dopa and OR-486 it appears that catecholamines particularly dopamine rather than cholinergic modulation is more important in adenosine induced catatonia. The motor dysfunction (catatonia) could be easily assessed using rota-rod test apparatus in mice.

Adenosine is known to be present in the CNS. Adeno­sine receptors namely A2A are present in high concen­tration in striatum, nucleus accumbens and olfactory tubercule (regions which are rich in dopamine) '. A2A and dopamine D2 receptors , which are co-localized in subpopulation of striatal projection neurons, the GABAergic output neurons of striatopallidal pathway have shown to be interacting antagonistically on dif­ferent levels. The A2A receptor agonists are reported to reduce the affinity for D2 receptors thus modulate D2 function2

.3 .

Adenosine modulates the release of variety of neu­rotransmitters both in vivo and in vitro. Adenosine has been shown to affect the release of norepinephrine, GABA, dopamine serotonin, acetylcholine, histamine, aspartate and glutamate. Although the mechanism by which adenosine affects neurotransmission is not es­tablished it has been generally accepted that adeno­sine acts through presynaptic modulation of neuro­transmitter release4

•5

.

The striatopallidal neuronal function seems to be mainly regulated not only by Au, receptors at the postsynaptic levels by direct antagonistic A2A-D2 and D2-A2A receptor-receptor interactions) but also at the presynaptic levels by an A2A receptor mediated regu­lation of acetylcholine release and, possibly, of GABA release. The evidence that stimulation of A2A receptors most probably localized in the striatal cho-

*Correspondent author : E-mail: Skpu@yahoo.com Fax : 91-0172-541142

linergic intemeurons has been reported to induce ace­tylcholine release in striatal synaptosomal prepara­tions6

•7 supports this.

With this background the present work was carried out to investigate adenosine-dopamine and adenosine­cholinergic interactions. Various models to test the motor function, including the rota-rod apparatus was used as an approach for the evaluation of catatonic moments in animals and the fall-off time in this test was compared with the other existing model of cata­tonia namely the bar immobility tests.

Materials and Methods Animals-Laka mice of either sex (20-30g), bred

in central animal house (CAR) of Panjab University, Chandigarh, maintained on a 12-h light and dark cy­cles were used in the study. Animals were housed un­der standard laboratory conditions, with free access to food and water. All behavioural experiments were carried out between 0900 and 1400 hrs. The experi­mental protocol was approved by Institutional Ani­mals Ethics Committee.

Assessment of catatonia-Using the following three-test procedures assessment of catatonia was done in animals.

Rota-rod test-Mice were subjected to motor function evaluation by placing them individually on rota-rod9

, which was adjusted to the speed of 21 r.p.m. The fall off time was recorded for each mouse and the longest time any animal was kept on the rod was 300 sec.

SINGH & KULKARNI: ADENOSINE IN DRUG-INDUCED CATATONIA 883

Bar test-In the bar test, front paw of the mice were gently placed on a horizontal metal bar with 2 mm diameter and placed 4 cm above ground level and the length of time the mouse maintained this ab­normal posture with at least one paw was measured8

.

The test was terminated when the paw of animal touched the ground or 180 sec had passed. If the ani­mal did not hold on to the bar after three attempts, it received the score of 0 seconds.

Drug treatment-The following drugs were used in the present study. Adenosine (Loba Chemicals, Mumbai, India) was dissolved with the aid of mini­mum quantity of hydrochloric acid, the volume was adjusted with distilled water, and finally pH was ad­justed to neutral. Perphenazine (Schering Co., Kenil­worth, NJ, USA) was dissolved with the aid of mini­mum quantity of hydrochloric acid, the volume was adjusted with distilled water, and finally pH was ad­justed to neutral. Scopolamine HBr (Merck & Co., Inc., NJ, USA) was dissolved in distilled water. L­dopa (Hi Media, Mumbai, India), carbidopa (Sun Pharmaceuticals, Mumbai, India), OR-486, (gift sam­ple from Prof. P.T Mannisto Finland) were suspended in 0.3% sodium CMC.

Treatment schedule-All the drugs were adminis­tered intraperitonially ip, in a constant volume of 1 ml per 100g of body weight of mice. Adenosine was ad­ministered 30 min prior to the behavioural assess­ment. Scopolamine, perphenazine, OR-486 was co­administered with adenosine respectively. Carbidopa was co-administered with adenosine as well as per­phenazine 15 min prior to the administration of L­dopa, behavioural assessment was done 1 hr and 2 hr of L-dopa administration, respectively.

Statistical analysis-The fall-off time in rota-rod test, the time spent in bar test were expressed as mean± SE. The data was analyzed using analysis of variance (ANOYA) followed by Dunnett's test by a statistical package STAT. In the test, the criterion for statistically significance was P<0.05.

Results Effect of perphenazine on fall-off time from the

rota-rod test and time spent on the bar in bar test­Perphenazine (1-5 mg/kg, ip) decreased the fall-off time and increased the time spent by the animal on the bar in a dose dependent manner. Significant differ­ences were observed at all the doses as compared to the non-treated control group (Figs 1 A & B).

Effect of adenosine on fall-off time and time spent on the bar in bar tests-Adenosine (10-100 mg/kg,

ip) decreased the fall-off time and increased the time spent by the animal on the bar in a dose dependent manner. Significant differences were observed at dose of (50-mg/kg, ip) and (100 mg/kg, ip) as compared to the non-treated control group (Figs 2 A & B).

Adenosine (l0-100 mg/kg, ip) when administered in combination with perphenazine (5 mg/kg, ip) de­creased the fall-off time and increased the time spent by the animal on bar in a dose dependent manner as compared to the perphenazine (5mg/kg, ip) alone treated group. Significant differences were observed at various dose of (25, 50 and 100 mg/kg, ip) of adenosine (Figs 3 A & B).

Effect of I-dopa and carbidopa combination on adenosine induced catatonia-L-dopa (100 mglkg, ip and 200 mg/kg, ip) and carbidopa (10 mg/kg, ip 20 mg/kg, ip) combination dose dependently increased the fall-off time when given in combination with adenosine (100 mg/kg, ip). Significant differences were found after 1 hr of I-dopa administration as compared to the control (adenosine 100 mg/kg, ip)

250 w-I/)

-II 200

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~ ~ 50 ~

w- 120 I/) -II 100

! 80 :z w Q. 60 I/)

w 40 ::;

i=

~ 20

~ 0

(A) Rota-rod test

CONTROL PPZ(1) PPZ(2.5) PPZ(5)

TREATMENT (mg /kg)

(8) Bar test

..

a

CONTROL PPZ(1) PPZ(2.5) PPZ(5)

TREA TMENT(mgIkg)

Fig. 1-Effect of perphenazine (1-5 mg/kg) on (A) fall-off time (8) time spent 011 bar by mice. *P<0.05 as compared untreated conrrol group. ** P < 0.05 as compared perphelluzine (I mg/kg) treated group. (ANOvA followed by Dunnett's test) a Animals did not hold on to the bar in three attempts. PPZ = perphenazine n=5

884 INDIAN J EX? BIOL, AUGUST 2002

alone treated group. Although there was no dose de­pendency after 2 hr but difference was stastically sig­nificant (Fig. 4). In the bar test this combination when administered in combination with adenosine (lOa

350 (A) Rota- rod test

CONTROL AOE(10) ADE (25) ADE(50) ADE (100)

TREATMENT (mgIkg)

ill' 120 (B) Bar test ... 1 100 •• f- 80 z w 60 0.. (/) w 40 :;; i= 20 z a a a ;1i 0 ~

CONTROL ADE (10) ADE(25) ADE(50) AOE(100)

TREATMENT(mglkg)

Fig. 2 - Effect of adenosine (10-100 mg/kg) on (A) fall off time, (B) time spent on bar by mice. * P < 0.05 as compared to the con­trol group. **p < 0.05 as compared to the adenosine 50 mg/kg (ANOY A followed by Dunnett's test). n=5 a Animal did not hold on to the bar in three attempts ADE = Adenosine

PPZ(5) (A)Rota-rod test

ADE(IOO)

~ E PPZ(S)ADE(IO) i=' Z w ~

~ PPZ(5}+ADE(2S)

0::: .... Ppz(S}+ADE(SO)

Ppz(S}+ADE(IOO)

0 10 20 30 40

MEAN FALL- OFF TIME (sec tSE)

mg/kg, ip) completely reversed adenosine-induced catatonia after 1 and 2 hr of I-dopa administration. The animals did not hold on to the bar in three at­tempts indicating a complete reversal (Table 1).

Effect of l-dopa and carbidopa combination on adenosine plus perphenazine induced catatonia­L-dopa (lOa mg/kg, ip and 200 mg/kg, ip) and carbi­dopa (10 mg/kg, ip and 20 mg/kg, ip) combination increased the fall-off time when administered along with the combination of adenosine (lOa mg/kg, ip) and perphenazine (5 mg/kg, ip) treated group. Al­though no dose dependency was observed but the effect was statistically significant after 1 and 2 hI' of I-dopa administration as compared to the control group, treated with combination of adenosine (lOa mg/kg,ip) plus perphenazine (5 mg/kg, ip) (Fig. 5). In the bar test I-dopa (lOa mg/kg, ip) and carbidopa (10 mg/kg, ip) combination significantly decreased the time spent by the animal on the bar when given along with the combination of adenosine (lOa mg/kg, ip) plus perphenazine (5 mg/kg, ip) after I hr of I-dopa administration as compared with the control group treated with the combination of adenosine (lOa mg/kg, ip) plus perphenazine (5 mg/kg, ip). There was a complete reversal seen after 2 hI' of I-dopa admini­stration with this dose. L-dopa (200 mg/kg, ip) and carbidopa (20 mg/kg, ip) combination when adminis­tered along with the combination of adenosine (lOa mg/kg, ip) plus perphenazine (5 mg/kg, ip) com­pletely reversed adenosine (lOa mg/kg, ip) plus per­phenazine (5 mg/kg, ip). Induced catatonia after 1 and 2 hr of I-dopa administration as the animal were not able to hold on to the rod in three attempts (Table 2).

(B)Bartest

*

*

~ *

50 0 50 100 150 200 MEAN TIME SPENT (sec tSEM)

Fig. 3-Effect of adenosine (10-100 mg/kg) on perphenazine (5 mg/kg) induced catatonia in (A) rota rod test (B) bar test. *P<0.05 as compared to control (perphenazine)(ANOY A followed by Dunnett's test). n = 5-10 P?Z = perphenazine ADE=adenosine

350

300 W II)

+I 250

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100 0 oJ oJ

~ 50 z ~

0 ~

SINGH & KULKARNI: ADENOSINE IN DRUG-INDUCED CATATONIA 885

OAdeno.lne(I00mg/kg)

.Adeno.Ine(IOOmg1kg}+L-dopa(lOCln'1Wkg)+C8I1lkIop8 (10 tnWkg)

BAdeno.Ine(I00mIVkll}+L-d0pa(200)+ carbidopa (20 rnWku)

Test time (hr)

2

Effect of OR-486 on adenosine-induced catatonia­OR-486 (30 mglkg, ip) significantly increased the fall-off time and when it was administered in combi­nation with adenosine (100 mglkg, ip) as compared to adenosine (100 mg/kg, ip) alone treated group (Fig. 6). In the bar test OR-486 (30 mglkg, ip) completely re­versed adenosine (100 mg/kg, i.p)- induced catatonia when it was administered in combination with adeno­sine (100 mglkg, ip) as compared with adenosine (100 mglkg, ip) treated group. The animals were not able to hold on to the rod in three attempts (Table 3).

Fig. 4-Effect of combination of L-dopa (100 mg/kg, 200 mglkg) and carbidopa (10 mg/kg, 20 mglkg) on adenosine in­duced catatonia in rota-rod test. *P<0.05 as compared to the control adenosine (100 mg/kg) at respective time inter­vals.(ANOY A followed by Dunnett's test). n = 6

Effect of scopolamine on adenosine-induced cata­tonia-Scopolamine (1 mglkg, ip and 2 mglkg, ip) increased the fall-off time in a dose dependent manner when administered in combination with adenosine (100 mglkg, ip). Significant difference was observed at dose of 2 mglkg, ip as compared to the adenosine (100 mglkg, ip) alone treated group (Fig. 7). In the bar test scopolamine (1 mglkg, ip) did not decrease the time spent by the animal on the bar when adminis-

Table I-Effect of combined treatment of L-dopa and carbidopa on adenosine induced catatonia in mice as tested on bar test

Treatment (mg/kg)

Adenosine (100)

Adenosine (100) after 1 hr

Bar test

Adenosine (100) +L-dopa (100) +carbidopa (10) after Ihr

Adenosine (100) +L-dopa (200) +carbidopa (20) after I hr

Adenosine (100) after 2 hr

Adenosine (100) +L-dopa (100) +carbidopa (10) after 2 hr

Adenosine (100) +L-dopa (200) +carbidopa (20) after 2 hr

** Animal did not hold the bar in three attempts.

Mean time spent (sec ± SE)

85.2 ±11.07

93.2±13.15 Reversed**

Reversed** 23.2 ± 5.2

Reversed**

Reversed**

Table 2-Effect of combined treatment of L-dopa and carbidopa on adenosine plus perphenazine induced catatonia in bar test

Treatment (mglkg)

Adenosine (100) + PPZ ( 5)

Adenosine (100) + PPZ (5) after 1 hr

Bar test

Adenosine (100)+ PPZ (5) +L-dopa (100) +carbidopa (10) after Ihr

Adenosine (100)+ PPZ (5) +L-dopa (200) +carbidopa (20) after Ihr Adenosine (100) + PPZ (5) after 2 hr

Adenosine (100) + PPZ (5) +L-dopa (100) +carbidopa (10) after 2hr

Adenosine (100)+ PPZ (5) +L-dopa (200) +carbidopa (20) after 2hr

Mean time spent (sec ± SE)

175.8±2.56

68.8±4.56

Reversed **

Reversed **

154.4±9.64

Reversed ** 92.6±2.6*

*P<0.05 as compared to the control group (adenosine 100 mg/kg + perphenazine 5mglkg) (ANOYA followed by Dunnett's test) **Animal did not hold onto the bar in three attempts.

886 INDIAN J EXP BIOL, AUGUST 2002

30

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5

Rota-rod te.t

C Adenosine(1 OOmg/kg)+per phenazine(5rn!VkIl)

• Adenosine(1 OOmg/kIl)+per phenazine(5rn!VkIl)+L­dopa(100mg/kg)+carbidop a (10 mg!kll)

"'Adenosine(100mg/k1l)+per phenazine(5mgIkg)+L­dopa(200)+catbidopa( 20 mglkll)

O+-~J-----~~--

1 2 Test time(hr)

Fig. S.-Effect of combination of L-dopa (100 mglkg, 200 mglkg) and carbidopa (10 mg/kg, 20 mglkg) on adenosine (100 mglkg) + perphenazine (Smg/kg) induced catatonia in rota-rod test. *p < 0.05 as compared to the control (adenosine +perphenazine l at respective time intervals.(ANOYA followed by Dunnett's test) n = S

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;J, u.

~ 50

::E 0 Adenosine (100) Adenosine (100)+

OR466(30)

TREA TMENT(mglkg)

Fig. 6-Effect of OR-486 on adenosine induced catatonia in rota­rod test. * P < O.OS as compared to the control (adenosine). (ANOY A followed by Dunnett's) n = S

tered in combination with adenosine (100 mg/kg, ip). Scopolamine (2 mg/kg, ip) when administered in combination with adenosine (l00 mg/kg, ip) signifi­cantly reversed adenosine (100 mg/kg, ip) induced catatonia. The animals did not hold on to the rod in three attempts (Table 4).

When scopolamine (2 mg/kg, ip) was administered to the animals receiving various doses of adenosine (10, 25, 50 mg/kg, ip) and perphenazine (5 mg/kg, i.p), there was an increase in the fall-off time signifi­cant effect being observed at doses of 10 and 25 mg of adenosine (Fig. 8 A). Although there was a de­crease in the time spent by the animal on the bar, the

90

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70 u 41 <II

W 60 :!: i= 50 u.. ... 0 40 ...J -' ~ 30 ll..

Z 20 i5

:!: 10

0 ADENOSINE (100) ADENOSINE ADENOSINE (100)

(1oo).seo (1) .seo (2)

TREATMENT(mglkg)

Fig. 7-Effect of scopolamine (1-2 mglkg) on adenosine induced catatonia in rota rod test. *P<0.05 as compared to control (adeno­sine) (ANOY A followed by Dunnett's test. seo = scopolamine n=S

Table 3-Effect of OR-486 on adenosine induced catatonia in bar test in mice

Bar test

Treatment (mglkg)

Adenosine (100) Adenosine (100) +OR·486 (30)

Mean time spent (sec ± SE)

8S.2±11.07 Reversed**

**Animal did not hold the bar in three attempts

Table 4-Effect of scopolamine on adenosine induced catatonia in bar test in mice

Bar test

Treatment (mglkg)

Adenosine (100)

Adenosine (100) +Scopolamine (I)

Adenosine (100) +Scopolamine (2)

Mean time spent (sec ± SE)

8S.2

88.46 ± 10.13

Reversed**

** Animal did not hold onto the bar in three attempts

response was not statistically significant as compared to their respective combinations per se (Fig 8 B).

Discussion Since adenosine A2A and dopamine D2 receptors

which are co-localized in a sub-population of striatal­projection neurons, the GABAergic output neurons of the striatopallidial pathway have been shown to inter­act antagonistically on different levels' A2A receptor agonists are reported to reduce the affinity of dopa­mine agonist for D2 receptors and thus modulate D2 function2,3.

The result of our study indicated that adenosine dose dependently (l0-100 mg/kg, ip) produced cata­tonia as indicated by rota-rod! bar tests . Adenosine

SINGH & KULKARNI: ADENOSINE IN DRUG-INDUCED CATATONIA 887

(A) Rota-rod test

ppz (5) +ade (10) 1--_ .....

~ ppz (5) +ade (10)+ sco (2)

~ ppz(5)+ade(2S) Z W :E ppz (5) +ade (25) + sco (2) 1-----....1

~ ~ ~S)+adeSO .-

ppz (5) +ade( 50) + sco( 2) t--....

(B) Bar test

*

o 10 20 30 o 00 100 100 200

MEAN F ALL- OFF TIME (sec± SE) MEAN TIME SPENT (sec ± SE)

Fig. 8-Effect of scopolamine (2 mg/kg) on perphenazine (5 mglkg)+adenosine (10-50 mglkg) induced catatonia in (A) rota-rod test (8) bar test. P <0.05 as compared to control (perphenazine plus adenosine). (ANOYA followed by Dunnett's test). ppz = perphenazine, ADE = adenosine sco= scopolamine n = 5

also potentiated the perphenazine-induced catatonia in a dose dependent manner. In accordance with the above explanation adenosine might have acted on the adenosine A2A receptors in the striatum co-localized with D2 receptors and thus causing a decreased affin­ity of dopamine towards the dopamine D2 receptors leading to motor dysfunction of the dopamine D2 re­ceptors. This would again lead to disinhibition of negative control of dopamine D2 receptors towards the indirect pathway leading to the increased activity of indirect pathway, and hence a decreased movement by indirect modulation of cortex as observed in the animals in the form of catatonia.

When adenosine is co-administered along with perphenazine, decreased function of the dopamine D2 receptors already existing due to the blockade by per­phenazine, adenosine potentiated this decreased func­tion by possibly reducing the affinity of dopamine towards the D2 receptors and causing severe catatonic effect.

The striatopallidal neuronal function seems to be mainly regulated not only by A2A receptors at the postsynaptic levels (by direct antagonistic A2A-D2 re­ceptor-receptor interactions) but also at the pre synap­tic level by an A2A receptor mediated regulation of acetylcholine release. The evidence that stimulation of A2A receptors, most probably localized on the striatal cholinergic interneurons, has been reported to induce acetylcholine release in striatal synaptosomal prepara­tions supports this6

: In accordance with the above ex­planation scopolamine might have acted on post-

synaptic muscaranic receptors, co-localized along with dopamine O2 and adenosine A2 receptors in the striatopallidal pathway in basal ganglia. The blockade of postsynaptic muscaranic receptors would have at­tenuated the effects due to increased pre-synaptic ace­tylcholine release, resulting from the stimulation of A2 receptors by adenosine.

The mechanism by which adenosine affects neuro­transmission is not fully established although it is generally accepted that adenosine acts through pre­synaptic modulation of neurotransmitter release. Al receptors are located pre and post synaptically on cell bodies, and on axons where they mediate inhibition of the neurotransmission, by the inhibition of the neuro­transmitter release, hyperpolarizing neuronal mem­branes, reducing excitability and firing rate and alter­ing axonal neurotransmission4

• When the combination of I-dopa and a peripheral DOPA decarboxylase in­hibitor, carbidopa was administered with adenosine, a complete reversal of adenosine was observed. The reversal was far better as compared to scopolamine as well as OR-486 (central COMT inhibitor), as indi­cated by both bar and rota-rod test. L-dopa when given in combination with peripheral DOPA decar­boxylase inhibitor carbidopa gets decarboxylated into dopamine, thus increasing the levels of dopamine. These increased levels of dopamine would have re­stored the depleted levels of dopamine due to adeno­sine. OR-486, a centrally acting COMT inhibitor when administered along with the adenosine com­pletely reversed adenosine-induced catatonia. OR-486

888 INDIAN J EXP BIOL, AUGUST 2002

might have inhibited the metabolism of catechola­mines thus further increasing the levels of dopamine. When it was administered along with adenosine it completely reversed the adenosine effect. Thus this study confirms the potent adenosine-catecholamine, particularly adenosine-dopamine interactions. This study further gives clues to the use of adenosine A2A

receptor antagonist as newer therapeutic agents in drug therapy of Parkinson's disease 13 .

Acknowledgement The authors appreciate Prof. P.T.Mannisto of Uni­

versity of Kupio, Kupio, Finland for generously providing COMT inhibitors for the study.

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receptors. Trends Pharmacol Sci, 17 (1996) 364. 2 Richarson P J, Kase H & Jenner P J, Adenosine A2A receptor

antagonist as new agents for the treatment of Parkinson's disease. Trends Pharmacol Sci, 18 (1997) 338.

3 Ferre S, Fredholm B B, Morelli M, Popoli P& Fuxe K, Adenosine-dopamine receptor- receptor interactions as an in­tegrative mechanism in the basal ganglia. Trends Neurosci, 20 (1997) 482.

4 Kulkarni S K & Thorat S N, Purinergic transmission: An up­date. Drugs Of Today, 26 (1990) 499.

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6 Ralevic V & Burnstock G, Receptors for Purines and Pyrimidines. Pharmacol Rev, 50 (1998) 415.

7 Jin S, Johansson B & Fredholm B B, Effects of adenosine AI and A2 receptor activation on electrically evoked dopamine and acetylcholine release from rat striatal slices. J Pharmacol ExpTher, 267 (1993) 801.

8 Costall B & Naylor R J, On catalepsy and catatonia and the predictability of the catalepsy test for neuroleptic activity. Psychopharmacology, 34 (1974b) 233.

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