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In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease.

Nabuurs RJ, Rutgers KS, Welling MM, Metaxas A, de Backer ME, Rotman M, Bacskai BJ, van Buchem MA, van der Maarel SM, van der Weerd L - PLoS ONE (2012)

Bottom Line: In vivo specificity for Aβ was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection.Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ.Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands. R.J.A.Nabuurs@LUMC.nl

ABSTRACT
This study investigated the in vivo properties of two heavy chain antibody fragments (V(H)H), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled V(H)H in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled V(H)H by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All V(H)H showed rapid renal clearance (10-20 min). Twenty-four hours post-injection (99m)Tc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for (99m)Tc-ni3A or DTPA((111)In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits.

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Specific in vivo Aβ binding after BBB disruption.After disruption of the BBB using a co-injection of 15% mannitol with pa2H-Alexa594 into the right carotid artery of an aged APP/PS1 mouse sacrificed 2 hrs post injection, amyloid plaques are clearly depicted in both hemispheres using a Thioflavin T (ThT) staining (A), while the pa2H-Alexa594 signal is only detected in the right hemisphere (B). More careful examination shows all Alexa594 signal colocalizes with ThT in the right hemisphere, while in the left only some autofluorescense can be detected. Furthermore, immunofluorescense anti-Aβ staining of the plaques using Alexa488 within the left hemisphere (C) results only in green signal, while within the right hemisphere (D) the red signal from pa2H-Alexa594 nicely colocalizes within the plaques. Experiments performed in a similar setting but sacrificed 24 hrs post-injection, showed similar results with pa2H-Alexa594 still nicely corresponding to the green labeling of the anti-Aβ staining within the right hemisphere (E).
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pone-0038284-g004: Specific in vivo Aβ binding after BBB disruption.After disruption of the BBB using a co-injection of 15% mannitol with pa2H-Alexa594 into the right carotid artery of an aged APP/PS1 mouse sacrificed 2 hrs post injection, amyloid plaques are clearly depicted in both hemispheres using a Thioflavin T (ThT) staining (A), while the pa2H-Alexa594 signal is only detected in the right hemisphere (B). More careful examination shows all Alexa594 signal colocalizes with ThT in the right hemisphere, while in the left only some autofluorescense can be detected. Furthermore, immunofluorescense anti-Aβ staining of the plaques using Alexa488 within the left hemisphere (C) results only in green signal, while within the right hemisphere (D) the red signal from pa2H-Alexa594 nicely colocalizes within the plaques. Experiments performed in a similar setting but sacrificed 24 hrs post-injection, showed similar results with pa2H-Alexa594 still nicely corresponding to the green labeling of the anti-Aβ staining within the right hemisphere (E).

Mentions: Based on the above findings, co-injections of pa2H-Alexa594 with mannitol were done in the right carotid artery to selectively open the BBB in the ipsilateral hemisphere to study the in vivo characteristics throughout the brain. Two hours post-injection, fluorescence was detected in the right hemisphere, co-localizing with Aβ. (Figure 4) Even within the deeper brain structures, no nonspecific binding was observed. Aβ related fluorescent signal remained detectable for at least 24 hours post-injection. Without BBB disruption or within wildtype littermates, no apparent Aβ labeling could be detected.


In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease.

Nabuurs RJ, Rutgers KS, Welling MM, Metaxas A, de Backer ME, Rotman M, Bacskai BJ, van Buchem MA, van der Maarel SM, van der Weerd L - PLoS ONE (2012)

Specific in vivo Aβ binding after BBB disruption.After disruption of the BBB using a co-injection of 15% mannitol with pa2H-Alexa594 into the right carotid artery of an aged APP/PS1 mouse sacrificed 2 hrs post injection, amyloid plaques are clearly depicted in both hemispheres using a Thioflavin T (ThT) staining (A), while the pa2H-Alexa594 signal is only detected in the right hemisphere (B). More careful examination shows all Alexa594 signal colocalizes with ThT in the right hemisphere, while in the left only some autofluorescense can be detected. Furthermore, immunofluorescense anti-Aβ staining of the plaques using Alexa488 within the left hemisphere (C) results only in green signal, while within the right hemisphere (D) the red signal from pa2H-Alexa594 nicely colocalizes within the plaques. Experiments performed in a similar setting but sacrificed 24 hrs post-injection, showed similar results with pa2H-Alexa594 still nicely corresponding to the green labeling of the anti-Aβ staining within the right hemisphere (E).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366949&req=5

pone-0038284-g004: Specific in vivo Aβ binding after BBB disruption.After disruption of the BBB using a co-injection of 15% mannitol with pa2H-Alexa594 into the right carotid artery of an aged APP/PS1 mouse sacrificed 2 hrs post injection, amyloid plaques are clearly depicted in both hemispheres using a Thioflavin T (ThT) staining (A), while the pa2H-Alexa594 signal is only detected in the right hemisphere (B). More careful examination shows all Alexa594 signal colocalizes with ThT in the right hemisphere, while in the left only some autofluorescense can be detected. Furthermore, immunofluorescense anti-Aβ staining of the plaques using Alexa488 within the left hemisphere (C) results only in green signal, while within the right hemisphere (D) the red signal from pa2H-Alexa594 nicely colocalizes within the plaques. Experiments performed in a similar setting but sacrificed 24 hrs post-injection, showed similar results with pa2H-Alexa594 still nicely corresponding to the green labeling of the anti-Aβ staining within the right hemisphere (E).
Mentions: Based on the above findings, co-injections of pa2H-Alexa594 with mannitol were done in the right carotid artery to selectively open the BBB in the ipsilateral hemisphere to study the in vivo characteristics throughout the brain. Two hours post-injection, fluorescence was detected in the right hemisphere, co-localizing with Aβ. (Figure 4) Even within the deeper brain structures, no nonspecific binding was observed. Aβ related fluorescent signal remained detectable for at least 24 hours post-injection. Without BBB disruption or within wildtype littermates, no apparent Aβ labeling could be detected.

Bottom Line: In vivo specificity for Aβ was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection.Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ.Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands. R.J.A.Nabuurs@LUMC.nl

ABSTRACT
This study investigated the in vivo properties of two heavy chain antibody fragments (V(H)H), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled V(H)H in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled V(H)H by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All V(H)H showed rapid renal clearance (10-20 min). Twenty-four hours post-injection (99m)Tc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for (99m)Tc-ni3A or DTPA((111)In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits.

Show MeSH
Related in: MedlinePlus