Effetti neurologici e
neurocomportamentali
Gemma Calamandrei
Reparto di Neurotossicologia e neuroendocrinologia
Dip Biologia cellulare e Neuroscienze
Istituto Superiore di Sanità
FOCUS OF OUR RESEARCH ACTIVITIES ARE
NEURODEVELOPMENTAL DISORDERS:
disabilities in brain functioning that affect a child’s
behaviour, memory, or ability to learn.
learn
They include mental retardation,
learning disabilities, ADHD, autism and autism
spectrum disorders (ASD)
Gene anomalies: 5%
Exposure to known teratogens (FAS, FACS): 30%
Obstetric complications: 15%
Psychosocial factors: 15-20%
Unknown etiology: 30-40%
Neurodevelopmental disorders:
polygenic and multifactorial etiology
• Environment act on (vulnerable) genes directly or
through epigenetic mechanisms
thus modulating/altering the cascade of events leading from gene
expression to establishment of brain circuitries
The brain has a protracted organogenesis and matures
from conception to adolescence
Formazione del Tubo Neurale
Si organizza in una serie di curve
e convoluzioni. Dopo circa 5 settimane
dal concepimento queste curve possono
essere identificate come le principali
componenti del cervello dei mammiferi
Mitosi/Proliferazione
Migrazione
Differenziazione
Aggregazione
Sinaptogenesi
Morte neuronale
Riorganizzazione
sinaptica
Mielinizzazione
Dall’eta neonatale
alla tarda adolescenza
Sequenza dei processi di maturazione
durante lo sviluppo fetale e postnatale
Postnatal development of neuronal connections in the cerebral cortex
1 month
2 months
2 years
Role of neurotrophic factors and steroid/thyroid hormones
Myelination
Plasticity = Vulnerability
Esposizione a un agente neurotossico a dosi
moderate o elevate in una fase precoce dello
sviluppo del SNC:
Acido valproico,
anticonvulsivante
Malformazione
= (i.e. spina bifida)
Scenario reale: esposizione protratta e a basse dosi
durante tutto il corso dello sviluppo pre e postnatale
perturbazione di molteplici processi maturativi
nel sistema nervoso, endocrino, immunitario
I processiQUANDO
maturativi COMINCIA
del cervello si IL
accompagnano
alla progressiva comparsa
COMPORTAMENTO?
dei comportamenti
ogni perturbazione
della sequenza di maturazione
Il feto complessi:
presenta attività
motorie diversificate
può determinare alterazioni
del comportamento
che preparano
alla vita postnatale
Progressiva maturazione dell’integrazione motoria e di
capacità cognitive complesse nei primi tre anni di vita
• Riconoscimento di volti familiari
• Riconoscimento di oggetti e manipolazione
• Sviluppo del linguaggio
• Gioco sociale (3° anno di vita)
• Lettura, scrittura, acquisizione
vocabolario
Xenobiotics
affecting
development in children
neuropsychological
¾Anticonvulsants
¾Alcohol
¾Nicotine
¾Metals: lead and methylmercury
¾Drugs of abuse (cocaine, heroin, etc.)
¾Polychlorobyphenils (PCB) and dioxins
Factors affecting neuropsychological development
¾prenatal stress
¾obstetric complications (asphyxia)
¾maternal infection
¾malnutrition
¾radiations
• Nella maggioranza dei casi, l’esposizione ad
agenti neurotossici in corso di sviluppo non
induce malformazioni cerebrali evidenti, ma
danni “sottili” che provocano alterazioni del
comportamento, che si manifestano in una fase
di sviluppo successiva, fino all’età adulta
L’ORGANISMO CRESCE NEL
PROPRIO DEFICIT!
Lo sviluppo neurocomportamentale normale o patologico
viene studiato nei modelli animali: roditori altriciali
Le sequenza di maturazione del SNC è simile a quella del feto umano
Gravidanza (circa 18-20 giorni)
Sviluppo postnatale estremamente rapido (circa 3 settimane) durante il
quale maturano progressivamente funzioni sensoriali e motorie assieme a
capacità di apprendimento associativo
Fase adolescenziale/prepuberale, contrassegnata dalla maturazione di
comportamenti sociali/esplorativi (21-45 giorni)
Maturità sessuale (60 giorni) e maturazione dei pattern comportamentali
specie/specifici e sessualmente dimorfici (comportamento parentale,
agonistico, ecc.) nonchè di capacità di apprendimento e memoria in test
complessi
Differenze nella morfologia del cervello
ma analoghi meccanismi di funzionamento
a livello delle connessioni tra neuroni
Tappe di maturazione simili
Brazelton battery: a neuromotor scale to assess
sensorimotor development in human new borns
Fox Scales for laboratory rodents:
assessment of sensorimotor development in rodents
a) RIGHTING REFLEX, pup returns to its feet when placed on its back;
b) CLIFF AVERSION, pup withdraws from the edge of a flat surface
when its snout and forepaws are placed over the cliff;
c) FORELIMB AND HINDLIMB STICK GRASP REFLEX, pup grasps a
toothpick when it is touched to the palm of each paw;
d) STRONG AND WEAK TACTILE STIMULATION TESTS, a headturning
response is triggered by the
application of tactile stimuli in
the perioral area on both sides
of the head;
e) POLE GRASPING, pup grips a
wooden pencil with its forepaws;
NEUROBEHAVIORAL ENDPOINTS OF POSTNATAL
SENSORY AND MOTOR DEVELOPMENT
g) VIBRISSA PLACING REFLEX, pup places its forepaw on an object
stroked across its vibrissae;
h) AUDITORY STARTLE RESPONSE,
pup shows a whole-body startle
response when a loud clap of the hands occurs less then 10 cm away;
f) LEVEL OR VERTICAL SCREEN TEST, pup
holds onto a wire mesh (5 x 5 mm) when
dragged across it horizontally or vertically by
the tail;
g) SCREEN CLIMBING TEST, pup dimbs up the
vertical screen using both fore- and hindpaws;
Spontaneous motor behaviour
• Locomotion
• Probing (search for the nipple) and head
raising
• Rolling
• Face-washing
• Twitching (abrupt movements of the whole body)
• Wall-climbing
Spontaneous activity and exploration
OPEN FIELD TEST (2):
- rearing (wall rearing and non-supported)
- grooming
- exploratory sniffing
- freezing
- lying still (without freezing)
- thigmotaxis (time spent in contact with the walls)
- stereotypes (i.e., increase to above a zero or very low control level of
acts such as face washing, gnawing, circling, jamping, head scanning,
and focused sniffing)
- digging and push digging (if the floor is covered with bedding material)
- urination and defecation
Social behaviours
- the investigation of social behavior in animals presents several concerns
that are not usually present when studying individual subjects
- the assessment of social behavior profile at successive developmental
phases is important since it is markedly age-dependent
- sexually dimorphic
- two kinds of social interactions during development:
a) mother-offspring
b) between individuals of same age
Automated data collection is impractical at best and
usually necessitate the use of observational (i.e.,
ethological) methodologies.
Spatial learning and memory
RADIAL MAZE
MORRIS WATER MAZE
hidden platform
before learning
sessions
after learning session
Assessment of behavioural flexibility
Radiation-induced brain injury
Delayed cognitive effects at adulthood in humans treated for brain tumors
Neuroanatomical Targets of Radiation-Induced Cognitive
Impairment not fully clarified yet!
The effects in children treated for brain tumors are
significant and consist in reduced IQ and lower school
rating.
Learning impairment and brain
alterations in rodent models
Brown Norway rats were irradiated with a total 40
Gy dose of fWBI delivered as 5 Gy fractions,
twice/week for 4 weeks.
Diffusion tensor image of a rat brain
color-coded to show the predominant
direction of diffusion in various brain
regions; blue indicates diffusion between
anterior (A) and posterior (P), red
indicates flow between left (L) and right
(R), and green indicates flow between
superior (S) and inferior (I). Adapted from
Robbins et al. (2012)
Mechanisms of brain injury by radiation: the
contribution of animal models
Vascular endothelial cells, oligodendrocytes, astrocytes,
microglia, and neurons: passive bystanders that merely die
from radiation damage, or active participants?
Orchestrated response of several cell types to radiation
injury: intervention to prevent cognitive impairment require
the detection of subtle molecular, cellular, and
microanatomic changes in the brain
Mechanisms underlying the
cognitive/behavioural effects of radiation
™Increased oxidative stress
™Increased apoptosis
™Reduced hippocampal neurogenesis
Young adult rats
irradiated with 10Gy
produced only 3% of
the new hippocampal
neurons
A decrease in hippocampal
neurogenesis has been
correlated with deficits in
hippocampal-dependent
spatial learning and memory
at 3 months after a single 5 Gy
dose of WBI to 21-day-old
mice (Rola et al., 2004)
Examination of
pro-inflammatory
cytokine gene
transcripts in the
brain
demonstrated
that gamma
radiation 2 Gy
increased
hippocampal
TNF-α
expression as
early as 4 h postirradiation
Hippocampal neurogenesis
the major target of low dose radiations
•
•
•
•
the process of neurogenesis continues throughout life in all mammals
including humans
dentate neurogenesis and behavioral performance are positively correlated in
animals
depletion of cells in the SGZ by ionizing irradiation has been hypothesized to
contribute to the cognitive impairments seen after such treatment
Plasticity: physiological reduction of neurogenesis with aging
Effects of radiations on cell proliferation
in the SVG of rats
Dose response curves for
apoptosis of wild-type (WT)
C57BL/6 mice (solid circles, a)
and p53 null mice (open
circles, a) were obtained at the
peak time of apoptosis in the
mouse, 12 h after exposure.
The steeper portion of the WT
response, between 0–2 Gy,
was dominated by the death of
actively proliferating cells (dark
bars, b), whereas in the
shallower portion (2–10 Gy),
there were increasing losses
of immature neurons (light
bars, b).Andres-Mach et al.,
Cell Tissue Res 2007
Effects of irradiation during
development
‰ Several papers on rats irradiated with doses ranging from 10 to 13 Gy
(1987-1990): reduced hipocampal nurogenesis and behavioural impairment
‰ Otake, M., Schull, W.J., 1998. Radiation-related brain damage and growth
retardation among the prenatally exposed atomic bomb survivors. Int. J.
Radiat. Biol. 74, 159–171.
‰ Fuss et al.,2000: Full Scale IQ (FSIQ) data, representing 1,938 children,
were derived from 36 publications and analyzed as to radiation dose,
irradiated volume, and age. The collected data suggest that whole brain
irradiation doses of 18 and 24 Gy have no major impact on intellectual
outcome in children older than age 6, but may cause impairment in younger
children. Doses > 24 Gy comprise a substantial risk for FSIQ decline, even
in older children.
‰ Sienkiewicz et al.(1999) showed that mice, prenatally exposed to 1 Gy of Xradiation at gestation day 18, were severely impaired in the performance of
learning and memory tasks.
‰ Effects significant at 0.2 Gy with a single irradiation in utero in mice!
Uno studio epidemiologico svedese sugli effetti neuropsicologici
delle base dosi
….doses below 0.50 Gy are assumed
to result in more subtle cognitive
dysfunctions .
Such cognitive changes as result of
low-dose exposure have been
observed in both pediatric and adult
patients and are often manifested as
deficits in the hippocampal-dependent
functions
of learning, memory, and spatial
information processing
The need for more research
•
•
Health risks involve not only neoplastic diseases but also somatic
mutations that may contribute to other illnesses (including birth
defects and ocular maladies) and heritable mutations that may
increase the risk of diseases in future generations. Unfortunately,
general statements on the health risks of low-dose radiation are
usually made by the analysis of data on the risk of cancer alone
Although the immediate molecular radiation response is reasonably
well understood, a more expanded and detailed analysis about
potential molecular pathways involved in the radiation response of
the developing brain is still required.
In vivo models
• Assessment of dose-response effects
• Identification of critical phases of vulnerability
• Mechanistic studies
• Evaluation of long-term effects
• Validation of biomarkers
A RESEARCH STRATEGY:
BEHAVIORAL CHARACTERIZATION OF RODENT MODELS OF LOWDOSE RADIATION EXPOSURE in parallel with BIOMARKERS of CNS
functioning;
IDENTIFICATION OF CRITICAL PERIOD OF VULNERABILITY;
TRANSLATION TO CLINICAL STUDIES
S coordination
Motor
e
s
n
Learning and memory
Water maze
Anxiety and exploration
What is to be considered safe for the effects of Low Dose irradiations?
As for Mercury: Declining Threshold of Harm following experimental
studies on animals and collection of clinical evidences
(micrograms/kg/day Hg)
DAILY INTAKE
100
Level associated with
harmful effect
Regulatory standard
(maximum safe exposure or high
end exposure from allowed fish
contamination)
10
1
FDA
WHO
ATSDR
0.1
EPA
0.01
1970
1980
1990
YEAR
2000