Thus, improved ways of determining the toxicity, such as the neurotoxicity, of a therapeutic molecule without requiring animal testing are needed. Taboo Uncensored animal lesbian sex Gif ! Share There were also two drawings and one computer-generated image depicting children in sexual poses and 131 images of extreme pornography showing women in lesbian sex gif acts with animals. The methods can be used in vitro or in silico to screen molecules before administration to laboratory animals or the methods can be used in vivo in laboratory animals. E39. The method of embodiment 38, wherein the molecule exhibits an in vivo tolerability score of 0 to 4 in each of the tolerability category. 14. The method of claim 1, further comprising measuring a behavioral test score of the molecule. A method of testing or determining in vivo acute neurotoxicity of a molecule comprising measuring calcium oscillations in vitro in neuronal cells which are in contact with the molecule.



The present disclosure provides a method of testing or determining in vivo acute neurotoxicity of a molecule comprising measuring oscillations in intracellular free calcium concentration ("calcium oscillations") in vitro in neuronal cells which are in contact with the molecule. E45. The method of any one of embodiments 1 to 44, further comprising measuring tubulin intensity of the molecule in a culture of neuronal cells. The above methods of the present invention can further comprise measuring tubulin intensity in a culture of neuronal cells, expression of a target protein, or behavioral performance of the molecule. In certain embodiments, the molecule comprises a protein, a peptide, a small molecule, a polynucleotide (e.g., an antisense oligonucleotide), or any combination thereof. E69. The method of any one of Embodiments 63 to 68, wherein the molecule comprises a small molecule, a polynucleotide, a protein, a peptide, or any combination thereof. E74. The method of any one of Embodiments 63 to 73, lesbian sex gif wherein the molecule is an antisense oligonucleotide. E56. The method of any one of embodiments 23 to 55, wherein the antisense oligonucleotide is a gapmer, a blockmer, a mixmer, or a wingmer. E87. In the method of any one of Embodiments 81 to 85, the improvement further comprising measuring an in vivo tolerability of the molecule.





E72. The method of any one of Embodiments 63 to 71, further comprising measuring in vivo tolerability of the molecule. The ASO and other oligomerss that behaved similarly, were shown to negatively impact central nervous system (CNS) network activity in vivo and electrophysiologic spontaneous neuronal activity in vitro (data not shown). One central concern when administering therapeutic molecules to a subject is the potential neurotoxicity of the molecules. Exposure to neurotoxic molecules can lead to damage to the brain and peripheral nervous system, causing long term physiological problems. E35. The method of embodiment 33, wherein the molecule is administered to the brain of the mammal. E88. In the method of Embodiment 87, wherein the in vivo tolerability is measured by administering the molecule to a mammal and grading the mammal's tolerability in a tolerability category. E36. A method of testing or determining in vivo tolerability of a molecule comprising administering the molecule to a mammal and grading the mammal's tolerability in a tolerability category.



11. The method of claim 1, further comprising measuring an in vivo tolerability of the molecule. In vivo tolerability scores were calculated based upon observations following a single intra-cerebroventricular (i.c.v.) administration of 100 .mu.g oligomers in mice or intra-thecal (i.t.) administration of 900 .mu.g oligomers or up to 1500 .mu.g in rats. E33. The method of any one of embodiments 1 to 32, further comprising measuring an in vivo tolerability of the molecule. E37. The method of any one of embodiments 34 to 36, wherein the tolerability category comprises at least one, at least two, at least three, at least four, or at least five tolerability categories. E73. The method of any one of Embodiments 63 to 72, further comprising measuring tublin intensity in a culture of neuronal cells. E14. The method of one of embodiments 1 to 13, further comprising administering the molecule to a subject in need of treatment of a disease or condition. E30. The method of any one of embodiments 23 to 29, wherein the antisense oligonucleotide modulates protein expression encoded by the target protein in the culture of the neuronal cells.