1. The document discusses various in vivo and in vitro models used to study Parkinson's disease (PD). In vivo models include antagonism of tremorine/oxotremorine-induced parkinsonism in mice, reserpine antagonism in mice, the MPTP model in monkeys, and circling behavior in nigrostriatal lesioned rats. In vitro methods include experiments using rat striatal slices and measuring dopamine stimulated adenyl cyclase activity. 2. The experimental autoimmune encephalomyelitis (EAE) model is described as the most widely used animal model for multiple sclerosis (MS). EAE is induced by immunizing mice with myelin antigens, causing an inflammatory de

1. Himanshu Yadav (201408006)
M Pharm.(Pharmacology)
SGT University

2. Parkinson's disease (PD)
 Parkinson's disease (PD) results primarily from the
death of dopaminergic neurons in the substantia
nigra.

3. IN VIVO MODELS
 Tremorine And Oxotremorine Antagonism
 MPTP Model In Monkeys
 Reserpine Antagonism
 Circling Behavior In Nigostriatal Lesioned Rats
 Elevated Body Swing Method
 Skilled Paw Reaching In Rats
 Stepping test in rats

4. IN VITRO METHODS
 1.Experiment using rat striatal slices
 2.Dopamine stimulated adenyl cyclase activity
 3.Radioligand binding studies for D1 & D2 dopamine
receptors
 4.Dopamine release from synaptosome.
 5. In vitro neuroprotective efficacy.

5. 1.TREMORINE AND
OXOTREMORINE ANTAGONISM:
PURPOSE AND RATIONALE
 The muscarinic agonists tremorine and oxotremorine
induce parkinsonism-like signs such as tremor, ataxia,
spasticity, salivation, lacrimation and hypothermia.
 These signs are antagonized by central anticholinergic
drugs.

6. PROCEDURE
 Group of 6-10 male NMRI mice (Swiss-type mouse, Naval
Medical Research Institute). (18-22g)
 Dosed orally with test/ standard (5mg/kg benzatropine
mesilate) 1h prior administration of 0.5mg/kg
oxotremorine (S.C)
 Before administration of oxotremorine (S.C) rectal
temperature is measured & taken as basal value.
 Rectal temperature is again measured after 1,2,3h after
oxotremorine inj.
 Tremor is scored after oxotremorine dosage at 10 sec
observation duration for every 15min interval for 1h and
scored.

7. Salivation and Lacrimation are also scored every15min and 30min after
oxotremorine inj.
SCORES ARE RECORDED AS FOLLOWS:
TREMOR, SALIVATION &
LACRIMATION SCORE
Absent 0
Slight 1
Medium 2
Severe 3
Test drug & standard should reduce the score.

8. CRITICAL ASSESSMENT OF THE
METHOD
 The oxotremorine antagonism has been proven to be a
reliable method for testing central anticholinergic
activity.
 The overt isomorphism between the animal model and
the symptoms of Parkinson’s disease recommend this
test for screening of anti-Parkinson drugs.
 However, the model measures only central
anticholinergic activity & cannot be used to screen
dopaminergic drugs.

9. 2. Reserpine antagonism
 PURPOSE AND RATIONALE
 Reserpine induces depletion of central catecholamine
stores.
 The sedative effect can be observed in mice shortly
after injection, followed by signs of eyelid ptosis,
hypokinesia, rigidity, catatonia, and immobility. These
phenomena can be antagonized by dopamine agonists.

10.  PURPOSE AND RATIONALE
 Reserpine induces depletion of central catecholamine
stores.
 The sedative effect can be observed in mice shortly
after injection, followed by signs of eyelid ptosis,
hypokinesia, rigidity, catatonia, and immobility. These
phenomena can be antagonized by dopamine agonists.

11. EVALUATION
 Locomotor activity and rearing & grooming scores of
drug treated animals are compared with controls
treated with reserpine and vehicle only by analysis of
variance.
 Locomotor activity of drug treated group must be
more than reserpine treated control.

12. 3.MPTP model in monkeys
 PURPOSE AND RATIONALE
 N-MPTP (N-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine)
 It has been shown to cause symptoms of Parkinson’s
disease in exposed individuals. When administered to
primates(monkeys as in this case) this compound
causes a partial destruction of basal ganglia (by ETC
blockade) and a syndrome that resembles Parkinson’s
disease.

13. 4.Circling behavior in
nigrostriatal lesioned rats
 PURPOSE AND RATIONALE
 Unilateral lesion of the dopaminergic nigrostriatal pathway in
the rat by the neurotoxin 6-hydroxydopamine (6OHDA) induces
hypersensitivity of the postsynaptic dopaminergic receptors in
the striatum of the lesioned side. The rats rotate in a circling
chamber in a direction towards the lesioned side (ipsilateral)
when an indirect acting compound such as amphetamine is
administered.
 But to the opposite direction (contralateral) when a direct acting
dopamine agonist, e.g., apomorphine, or the dopamine precursor
L-dopa is given. Therefore, this test can be used for the study of
central dopamine function and the evaluation of dopamine
antagonists and agonists, particularly the activity of novel
antiparkinsonian drugs.

14. 5. Elevated body
swing test
 PURPOSE AND RATIONALE
 Borlongan and Sanberg (1995) proposed the elevated body
swing test as a measure of asymmetrical motor behavior of
parkinsonian animals in a drug-free state.
 The total number of swings made to each side is divided by
the overall total number of swings made to both sides to
get percentages of left and right swings. The criterion of
biased swing is set at 70% or higher.
 6-OHDA-lesioned rats exhibit right biased swings of 70%
or higher compared to normal rats.

15. PURPOSE AND RATIONALE
 The term “staircase test” mentioned in this context, has nothing to do with
the staircase test for evaluating anxiolytic activity in rats.
 Unilateral injection of 6-OHDA into the medial forebrain bundle results in
an impairment of paw reaching on both sides which can be ameliorated by
drug treatment or transplantation of a nigral cell suspension.
 The number of Saccharin pellets eaten during the test period indicates the
rat’s success in grasping and retrieving the pellets; the number of steps from
which pellets have been removed provides an index of the attempts to reach
the food and how far the rat can reach; the number of missed pellets
remaining at the end of the test on the floor of the side compartment
indicates a lack of sensorimotor coordination in grasping and retrieving the
pellets
 The apparatus has been developed after earlier studies of motor cortex,
nigrostriatal dopamine and caudate-putamen to skilled forelimb use in the
rat which is impaired in PD.

16. IN VITRO METHODS
1.EXPERIMENT USING RAT STAITAL SLICES
2. DOPAMINE STUMILATED ADENYLY CYCLASE
ACTIVITY

17. Screening model for MS
 Invitro Methods –
 Microglia
 Oligodendrocytes
 Astrocytes
 Invivo Methods -
 The experimental autoimmune/allergic encephalomyelitis (EAE)
 Viral induced models, mainly Theiler's murine encephalomyelitis virus
(TMEV) infection and consequential chronic demyelination.
 Toxin-induced models of demyelination, such as the cuprizone and the lyso-
phosphatidylcholine (lysolecithin) mod

18. In vivo Methods
The experimental autoimmune/allergic encephalomyelitis (EAE)
 The most-studied animal model of MS is the experimental
autoimmune encephalomyelitis (EAE), in which autoimmunity to CNS
components is induced in susceptible mice through immunization
with self-antigens derived from basic myelin protein.
 EAE may be induced in mice with different genetic backgrounds, such
as SJL/J, C57BL/6 and NOD, through either active immunization with
protein or peptide, or by passive transfer of encephalitogenic T cells.
 The relevant immunogen is derived from self-CNS proteins such as
myelin basic protein (MBP), proteolipid protein (PLP) or myelin
oligodendrocyte glycoprotein (MOG).

19. Procedure
 Active EAE relies on CNS reactive T cell that are induced following
immunization by an auto antigen.
 Emulsified in an adjuvant , EAE is induced by transferring auto reactive T cell
to recipient animal .
 Adjuvants which are used contains killed mycobacteria that elicit T cell
responses as well as antibody production due to innate immune activation .
 Immunization of SJL/J mice with the immunodominant epitope of PLP
(PLP139–151) induces a relapsing–remitting (RR) disease course, while disease
induced by the immunodominant MOG(35–55) peptide in C57BL6/J mice is of
chronic nature .

20.  MOG(35–55) was able to induce direct CNS autoimmunity, and
accompanied administration of pertussis toxin (PT) was able to
enhance disease onset and severity.
 Pathogenesis of EAE

 Substantial evidence from MS subjects indicates that CD8+ T
cells play a key role in the pathogenesis of the disease .CD8+T
cells have been found more represent than CD4+T cells in acute
and chronic CNS lesions of MS patients.
 EAE in mice is characterized by an ascending paralysis beginning
at the tail followed by limb and forelimb paralysis, assessed by
using a 5-points scale.

21.  MOG(35–55) was able to induce direct CNS autoimmunity,
and accompanied administration of pertussis toxin (PT)
was able to enhance disease onset and severity.
 Pathogenesis of EAE

 Substantial evidence from MS subjects indicates that CD8+
T cells play a key role in the pathogenesis of the disease
.CD8+T cells have been found more represent than CD4+T
cells in acute and chronic CNS lesions of MS patients.
 EAE in mice is characterized by an ascending paralysis
beginning at the tail followed by limb and forelimb
paralysis, assessed by using a 5-points scale.

22.  Immunotherapies specifically targeting CD4+ T cells failed to
show significant clinical benefit in MS course, whereas therapies
that affect all leukocytes can improve disease progression.
 Theiler's murine encephalomyelitis virus (TMEV) infection
 Viral infections of the CNS can induce demyelination in mice
and the best studied are the picornavirus, such as Theiler's
murine encephalomyelitis virus (TMEV).
 TMEV is divided into two subgroups, based on the ability to
cause disease in the CNS :-

23.  GDVII
 TO
 GDVII subgroup - Highly neurovirulent for mice, as it induces death within 1
to 2 weeks. GDVII virus predominantly infects neurons and dying neurons
display chromatin condensation and apoptotic (fragmented) nuclei
(karyorrhexis).
 TO subgroup -- The DA and BeAn strains of the TO subgroup induce acute
polioencephalomyelitis.
 In TMEV infection, axonal damage precedes demyelination (inside-out model)
and the distribution of damaged axons observed during the early phase
corresponds to regions, where subsequent inflammatory demyelination occurs
during the chronic phase .


24.  Toxin-induced models of MS
 Two most common agents utilized to induce demyelination are
 Cuprizone
 Lysolecithin.
 Cuprizone (biscyclohexanone-oxaldihydrazone) is a copper chelating reagent which,
supplemented to normal rodent chow, causes oligodendroglial cell death with
subsequent demyelination, together with a profound activation of astrocytes and
microglia.
 Specific targets of cuprizone in mice are mature oligodendrocytes, which fail to fulfill the
extensive metabolic demand and consequently undergo apoptosis, while the other cell
types are not


25.  affected. The main reason for the metabolic failure is copper
deficiency due to the copper chelation properties of cuprizone.
 Lysolecithin is an activator of phospholipase A2 which induces
focal areas of demyelination upon injection into the spinal cord
in several animals, including cat, rabbit, rat and mouse.
 Demyelination occurs due to primary toxic effects of detergent
on myelin sheaths, rather than to secondary effects on
oligodendrocytes.
 Infiltration and activation of macrophages and microglia begin
within hours after injection and last many days.

26. References
1) Parkinson's disease: mechanisms and models
2) William Dauer 1, Serge Przedborski
3) Viral models of multiple sclerosis:
neurodegeneration and demyelination in mice
infected with Theiler's virus
4) Miriam Mecha 1, Francisco J Carrillo-Salinas, Leyre
Mestre, Ana Feliú, Carmen Guaza